Bispecific molecules comprising an hiv-1 envelope targeting arm

ABSTRACT

The invention is directed to bispecific molecules comprising an HIV-1 envelope targeting arm and an arm targeting an effector cell, compositions comprising these bispecific molecule and methods of use. In certain aspects, the bispecific molecules of the present invention can bind to two different targets or epitopes on two different cells within the first epitope is expressed on a different cell type than the second epitope, such that the bispecific molecules can bring the two cells together. In certain aspects, the bispecific molecules of the present invention can bind to two different cells, wherein the bispecific molecules comprises an arm with the binding specificity of A32, 7B2, CH27, CH28 or CH44.

This application is a continuation of U.S. application Ser. No.15/514,420 filed Mar. 24, 2017 which is a U.S. National Stageapplication of PCT/US2015/053027, filed Sep. 29, 2015, which claims thebenefit of and priority to U.S. Ser. No. 62/056,834 filed Sep. 29, 2014,and U.S. Ser. No. 62/206,586 filed Aug. 18, 2015, the contents of eachof which are hereby incorporated by reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos. U19AI067854 and UM1 AI100645 awarded by the National Institutes of Health.The government has certain rights in the invention.

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosureas it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

All patents, patent applications and publications cited herein arehereby incorporated by reference in their entirety. The disclosure ofthese publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art as known to those skilled therein as of the date of theinvention described herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 29, 2020, isnamed 1234300_00344 US2_SL.txt and is 117,071 bytes in size.

TECHNICAL FIELD

The invention is directed to HIV-1 antibodies and bispecific moleculescomprising an HIV-1 binding domain and an effector cell binding domain,and their uses.

BACKGROUND

Highly Active Antiretroviral Therapy (HAART) has been effective inreducing the viral burden and ameliorating the effects of HIV-1infection in infected individuals. However, despite this therapy thevirus persists in the individual due to latent reservoir of HIV infectedcells which evade this treatment. Thus, there is a need for therapeuticagents for treatment of HIV-1 infected individuals, as well as agentsthat target virus infected cells and have to potential to reduce thelatent reservoir of HIV-1 infected cells.

SUMMARY OF THE INVENTION

The present invention is directed to bispecific molecules, e.g.covalently linked polypeptide chains to form antibodies, covalentdiabodies and/or covalent diabody molecules and their use in thetreatment of HIV-1. In certain aspects, the bispecific molecules of thepresent invention can bind to two different targets or epitopes on twodifferent cells wherein the first epitope is expressed on a differentcell type than the second epitope, such that the bispecific moleculescan bring the two cells together. In certain aspects, the bispecificmolecules of the present invention can bind to two different cells,wherein the bispecific molecules comprises an arm with the bindingspecificity of A32, 7B2, CH27, CH28 or CH44, which arm binds to theHIV-1 envelope expressed on a first cell, e.g. HIV infected cell, and asecond arm with the binding specificity for an epitope expressed on adifferent cell type than the first cell, such that the bispecificmolecules can bring the two cells together. In certain embodiment, thesecond cell is in effector cell which expresses CD3 or CD16.

In certain embodiments an antibody binds specifically to a particulartarget, even where the specific epitope may not be know, peptide orpolysaccharide (such as an antigen present on the surface of a pathogen,for example gp120, gp41, or CD3) and do not bind in a significant amountto other proteins or polysaccharides present in the sample or subject.Specific binding can be determined by methods known in the art. Variouscompetitive binding assays are known in the art. With reference to anantibody antigen complex, in certain embodiments specific binding of theantigen and antibody has a KD of less than about 10⁶ Molar, such as lessthan about 10⁶ Molar, 10⁷ Molar, 10⁸ Molar, 10⁹, or even less than about10¹⁰ Molar.

In certain aspects the invention provides bispecific moleculescomprising a first polypeptide chain and a second polypeptide chain,covalently bonded to one another, wherein:

-   -   (I) the first polypeptide chain comprises in the N- to        C-terminal direction:        -   (i) a domain (A) comprising a binding region of the light            chain variable domain of a first immunoglobulin (VL1) having            the binding specificity of the A32, 7B2, CH28, or envelope            antibody;        -   (ii) a domain (B) comprising a binding region of a heavy            chain variable domain of a second immunoglobulin (VH2)            specific for an epitope (2), wherein domains (A) and (B) are            separated from one another by a peptide linker 1; and        -   (iii) a domain (C) comprising a heterodimer promoting domain            including a K coil or E coil; wherein the heterodimer            promoting domain (C) and domain B are separated by a peptide            linker 2;    -   (II) the second polypeptide chain comprises in the N- to        C-terminal direction:        -   (i) a domain (D) comprising a binding region of a light            chain variable domain of the second immunoglobulin (VL2)            specific for the epitope (2);        -   (ii) a domain (E) comprising a binding region of a heavy            chain variable domain of the first immunoglobulin (VH1)            having the binding specificity of the A32, 7B2, CH28, or            CH44 HIV-1 antibody, wherein domains (D) and (E) are            separated from one another by a peptide linker 1; and        -   (iii) a domain (F) comprising a heterodimer promoting domain            including a K coil or E coil; wherein the heterodimer            promoting domain (F) and domain (E) are separated by a            peptide linker 2; and wherein:            the domains (A) and (B) do not associate with one another to            form an epitope binding site;            the domains (D) and (E) do not associate with one another to            form an epitope binding site; and            the domains (A) and (E) associate to form a binding site            that binds the HIV-1 envelope like A32, 7B2, CH28, or CH44            antibody (1); and the domains (B) and (D) associate to form            a binding site that binds the epitope (2).

In certain aspects the invention provides bispecific moleculescomprising a first polypeptide chain, a second polypeptide chain, and athird polypeptide chain, wherein some of the polypeptides are covalentlybonded (See FIG. 8), and wherein:

(I) the first polypeptide chain comprises in the N- to C-terminaldirection:

-   -   (i) a domain (A) comprising a binding region of the light chain        variable domain of a first immunoglobulin (VL1) having the        binding specificity of the A32, 7B2, CH28, or CH44 HIV-1        antibody;    -   (ii) a domain (B) comprising a binding region of a heavy chain        variable domain of a second immunoglobulin (VH2) specific for an        epitope (2), wherein domains (A) and (B) are separated from one        another by a peptide linker 1;    -   (iii) a domain (C) comprising a heterodimer promoting domain        including a K coil or E coil; wherein the heterodimer promoting        domain (C) and domain B are separated by a peptide linker 2;    -   (iv) a CH2-CH3 domain, wherein the CH2-CH3 domain and domain (C)        are separated by a peptide linker 3 or a spacer-linker 3;        (II) the second polypeptide chain comprises in the N- to        C-terminal direction:    -   (i) a domain (D) comprising a binding region of a light chain        variable domain of the second immunoglobulin (VL2) specific for        the epitope (2);    -   (ii) a domain (F) comprising a binding region of a heavy chain        variable domain of the first immunoglobulin (VH1) having the        binding specificity of the A32, 7B2, CH28, or CH44 HIV-1        antibody, wherein domains (D) and (E) are separated from one        another by a peptide linker 1;    -   (iii) a domain (F) comprising a heterodimer promoting domain        including a K coil or E coil; wherein the heterodimer promoting        domain (F) and domain (E) are separated by a peptide linker 2;        (III) the third polypeptide chain comprises in the N- to        C-terminal direction:    -   (i) a peptide linker 3,    -   (ii) a CH2-CH3 domain, and wherein:        the domains (A) and (B) do not associate with one another to        form an epitope binding site;        the domains (D) and (E) do not associate with one another to        form an epitope binding site;        the domains (A) and (E) associate to form a binding site that        binds the HIV-1 envelope lik A32, 7B2, CH28, or CH44 antibody        (I);        the domains (B) and (D) associate to form a binding site that        binds the epitope (2); and the CH2-CH3 domains of the first and        third polypeptide form an Fc chain.

A bispecific molecule comprising a first polypeptide chain, a secondpolypeptide chain, and a third polypeptide chain, wherein some of thepolypeptides are covalently bonded (See FIG. 8), and wherein:

(I) the first polypeptide chain comprises in the N- to C-terminaldirection:(i) a peptide linker 3 followed by a CH2-CH3 domain;(ii) a domain (A) comprising a binding region of the light chainvariable domain of a first immunoglobulin (VL1) having the bindingspecificity of the A32, 7B2, CH28, or CH44 HIV-1 antibody, wherein theCH2-CH3 domain and domain (A) are separated by a peptide linker 4;(iii) a domain (B) comprising a binding region of a heavy chain variabledomain of a second immunoglobulin (VH2) specific for an epitope (2),wherein domains (A) and (B) are separated from one another by a peptidelinker 1;(iv) a domain (C) comprising a heterodimer promoting domain including aK coil or F coil; wherein the heterodimer promoting domain (C) anddomain B are separated by a peptide linker 2;(II) the second polypeptide chain comprises in the N- to C-terminaldirection:(i) a domain (D) comprising a binding region of a light chain variabledomain of the second immunoglobulin (VL2) specific for the epitope (2);(ii) a domain (E) comprising a binding region of a heavy chain variabledomain of the first immunoglobulin (VH1) having the binding specificityof the A32, 7B2, CH28, or CH44 HIV-1 antibody, wherein domains (D) and(E) are separated from one another by a peptide linker 1;(iii) a domain (F) comprising a heterodimer promoting domain including aK coil or E coil; wherein the heterodimer promoting domain (F) anddomain (F) are separated by a peptide linker 2;(III) the third polypeptide chain comprises in the N- to C-terminaldirection:

-   -   (i) a peptide linker 3,    -   (ii) a CH2-CH3 domain, and wherein:        the domains (A) and (B) do not associate with one another to        form an epitope binding site;        the domains (D) and (E) do not associate with one another to        form an epitope binding site;        the domains (A) and (F) associate to form a binding site that        binds the HIV-1 envelope like A32, 7B2, CH28, or CH44 antibody        (1);        the domains (B) and (D) associate to form a binding site that        binds the epitope (2); and the CH2-CH3 domains of the first and        third polypeptide form an Fc chain.

In certain embodiments, the CH2-CH3 domain of polypeptide chain 1 is theof the “knob” design and the CH2-CH3 domain of the third polypeptidechain is of the “hole” design, or vice versa.

In certain embodiments, the epitope (2) is a CD3 epitope or a CD16epitope, In certain embodiments, the bispecific molecule binds HIVenvelope with the specificity of A32 antibody and also binds CD3. Incertain embodiments, the bispecific molecule binds HIV envelope with thespecificity of 7B2 antibody and also binds CD3. In certain embodiments,the bispecific molecule binds HIV envelope with the specificity of CH28antibody and also binds CD3. In certain embodiments, the bispecificmolecule binds HIV envelope with the specificity of CH44antibody andalso binds CD3. In certain embodiments, the bispecific molecule bindsHIV envelope with the specificity of A32 antibody and also binds CD16.In certain embodiments, the bispecific molecule binds HIV envelope withthe specificity of 7B2 antibody and also binds CD16. In certainembodiments, the bispecific molecule binds HIV envelope with thespecificity of CH28 antibody and also binds CD16. In certainembodiments, the bispecific molecule binds HIV envelope with thespecificity of CH44antibody and also binds CD16.

In certain embodiments, the domains (A) and (E) associate to form abinding site that binds the HIV-1 envelope with the binding specificityof the A32, 7B2, CH28, or CH44 antibody. In certain embodiments, thedomains (A) and (E) associate to form a binding site that binds the A32,7B2, CH27, CH28, or CH44 HIV-1 antibody epitope.

In certain embodiments, the domain (A) binding region of the A32immunoglobulin (VL1) comprises the VL-A32 CDR3, CDR2, and CDR1. Incertain embodiments, wherein the domain (E) binding region of the A32immunoglobulin (VH1) comprises the VH-A32 CDR3, CDR2, and CDR1. Incertain embodiments, the domain (A) binding region of the 7B2immunoglobulin (VL1) comprises the VL-7B2 CDR3, CDR2, and CDR1. Incertain embodiments, the domain (E) binding region of the 7B2immunoglobulin (VH1) comprises the VH-7B2 CDR3, CDR2, and CDR1. Incertain embodiments, the domain (A) binding region of the CH28immunoglobulin (VL1) comprises the VL-CH28 CDR3, CDR2, and CDR1. Incertain embodiments, the domain (E) binding region of the CH28immunoglobulin (VH1) comprises the VH-CH28 CDR3, CDR2, and CDR1. Incertain embodiments, the domain (A) binding region of the CH44immunoglobulin (VL1) comprises the VL-CH44 CDR3, CDR2, and CDR1. Incertain embodiments, the domain (E) binding region of the CH44immunoglobulin (VH1) comprises the VH-CH44 CDR3, CDR2, and CDR1.

In certain embodiments, the domain (A) comprises VL-A32, VL-7B2,VL-CH28, or VL-CH44. In certain embodiments, the domain (E) comprisesVH-A32, VH-7B2, VH-CH28, VH-CH44.

In certain embodiments, the first polypeptide comprises SEQ ID NO: 9,SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21. SEQ ID NO: 25, or SEQ IDNO: 44. In certain embodiments, the second polypeptide comprises SEQ IDNO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, orSEQ NO: 45. In certain embodiments, the bispecific molecule comprisesthe complementary second polypeptide, and wherein the second polypeptidecomprises SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ) NO: 23, SEQID NO: 27 or SEQ ID NO: 45.

In certain embodiments, the bispecific molecule comprises the firstpolypeptide of SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO:21, SEQ ID NO: 25, or SEQ ID NO: 44 and the second polypeptide of SEQ IDNO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, orSEQ ID NO: 45.

In certain embodiments, the bispecific molecule comprises the firstpolypeptide of SEQ ID NO: 9, and the complementary second polypeptide ofSEQ ID NO: 11. In certain embodiments, the bispecific molecule comprisesthe first polypeptide of SEQ ID NO: 13, and the complementary secondpolypeptide of SEQ ID NO: 15. In certain embodiments, the bispecificmolecule comprises the first polypeptide of SEQ ID NO: 17, and thecomplementary second polypeptide of SEQ ID NO: 19. In certainembodiments, the bispecific molecule comprises the first polypeptide ofSEQ ID NO: 21, and the complementary second polypeptide of SEQ ID NO:23. In certain embodiments, the bispecific molecule comprises the firstpolypeptide of SEQ ID NO: 25, and the complementary second polypeptideof SEQ ID NO: 27.

In certain embodiments, the bispecific molecule comprises consistingessentially of the first polypeptide of SEQ NO: 9. SEQ ID NO: 13, SEQ IDNO: 17, SEQ ID NO: 21, SEQ ID NO: 25, or SEQ ID NO: 44 and the secondpolypeptide of SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO:23, SEQ ID NO: 27, or SEQ ID NO: 45.

In certain embodiments, the bispecific molecule comprises consisting ofthe first polypeptide of SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQID NO: 21, SEQ NO: 25, or SEQ ID NO: 44 and the second polypeptide ofSEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO:27, or SEQ ID NO: 45.

In certain embodiments, the bispecific molecule comprises SEQ ID NO: 46,47 and 48. In certain embodiments, the bispecific molecule consistsessentially of SEQ ID NO: 46, 47 and 48. In certain embodiments, thebispecific molecule consists of SEQ ID NO: 46, 47 and 48. In certainembodiments, the first polypeptide of the bispecific molecule comprisesSEQ ID NO: 46, the second polypeptide of the bispecific moleculecomprises SEQ ID NO: 47, and the third polypeptide of the bispecificmolecule comprises SEQ ID NO: 48.

In certain aspects, the invention provides a composition comprising anyone of the bispecific molecules or any combination thereof. In certainembodiments, the composition comprises a composition comprising abispecific molecule comprising a first arm with the binding specificityof HIV-1 antibody A32, HIV-1 antibody 7B2, HIV-1 antibody CH28, HIV-1antibody CH44 and a second arm targeting CD3 or CD16. In certainembodiment, the bispecific molecule comprises an Fc portion or any othermodification which extends its serum half-life. In certain embodiments,the composition further comprises a second bispecific moleculecomprising a first arm with the binding specificity of the HIV-1antibody A32, HIV-1 antibody 7B2, HIV-1 antibody CH28, HIV-1 antibodyCH44 and a second arm targeting CD3 or CD16, wherein the first andsecond bispecific molecules are different.

In certain aspects, the invention provides a method to treat or preventHIV-1 infection in a subject in need thereof comprising administering tothe subject a composition comprising any one of the bispecific moleculesof the invention or a combination of any one of the bispecific moleculesin a therapeutically effective amount. In certain embodiments, themethods of claim further comprise administering a latency activatingagent. In some embodiments, the latency activating agent is vorinostat,romidepsin, panobinostat, disulfiram, JQ1, bryostatin, PMA, inonomycin,or any combination thereof.

In certain aspects, the invention provides nucleic acids comprisingnucleotides encoding the bispecific molecules of the invention. Incertain aspects, the invention provides a vector comprising nucleicacids comprising nucleotides encoding the bispecific molecules of theinvention. Provided are also compositions comprising a vector comprisinga nucleic acid encoding the bispecific molecules. In certain aspects theinvention provide a cell line comprising vectors or nucleic acidsencoding the bispecific molecules of the invention, wherein the vectorsencode polypeptide chains for expression of the bispecific molecules ofthe invention, e.g., polypeptide chain 1 and polypeptide chain 2, orpolypeptide chain 1, polypeptide chain 2 and polypeptide chain 3. Incertain embodiments, the vector is suitable for gene delivery andexpression. In certain embodiment, the vector is an adenoviral vector,an adeno associated virus based vector, or a combination thereof.

In certain aspects, the invention provides a bispecific moleculecomprising a polypeptide with a dual affinity retargeting reagent(DART), wherein the DART comprises a diabody molecule comprising a firstpolypeptide chain and a second polypeptide chain, covalently bonded toone another, wherein:

-   -   (A) the first polypeptide chain comprises:    -   (i) a domain (A) comprising a binding region of the light chain        variable domain of a first immunoglobulin (VL1) specific for the        first epitope (I); wherein the first VIA comprises, consists        essentially of, consists of the VL or VLCDR1, VLCDR2, and VLCDR3        from A32, 7B2, CH27, CH28, or CH44 HIV-1 antibody,    -   (ii) a domain (B) comprising a binding region of a heavy chain        variable domain of a second immunoglobulin (VH2) specific for a        second target, e.g an epitope (2), wherein domains (A) and (B)        are separated from one another by a peptide linker; and    -   (iii) a domain (C) comprising a heterodimer promoting domain;    -   (B) the second polypeptide chain comprises:    -   (i) a domain (D) comprising a binding region of a light chain        variable domain of the second immunoglobulin (VL2) specific for        the epitope (2);    -   (ii) a domain (E) comprising a binding region of a heavy chain        variable domain of the first immunoglobulin. (VH1) specific for        the first epitope (I); wherein the first VH1 comprises, consists        essentially of, consists of the VH or VHCDR1, VHCDR2, and VHCDR3        from A32, 7B2, CH27, CH28, or CH44 HIV-1 antibody, wherein        domains (D) and (E) are separated from one another by a peptide        linker, and    -   (iii) a domain (F) comprising a heterodimer promoting domain,        and    -   wherein:    -   the domains (A) and (B) do not associate with one another to        form an epitope binding site;    -   the domains (D) and (E) do not associate with one another to        form an epitope binding site;    -   the domains (A) and (E) associate to form a binding site that        binds the A32, 7B2, CH27, CH28, or CH44 HIV-1 antibody epitope        (1); the domains (B) and (D) associate to form a binding site        that binds the second target, e.g., epitope (2).

In certain embodiments, the invention provides bispecific molecules,wherein the HIV antibodies VH and VL domains, and the CD3 and CD16 VHand VL domains are in a different orientation. For example, in anon-limiting embodiment, the VL1 domain in polypeptide chain 1 is fromCD3, and VH2 domain is from an HIV envelope binding antibody. In thisembodiment, the VH1 domain of polypeptide 2 is from CD3, and VL2 domainis from is from an HIV envelope binding antibody.

In certain aspects, the invention provides a bispecific molecule capableof specific binding to HIV-1 envelope and to an epitope of CD3, whereinthe bispecific molecule comprises a first polypeptide chain and a secondpolypeptide chain, covalently bonded to one another, wherein:

-   -   A. the first polypeptide chain comprises, in the N-terminal to        C-terminal direction:        -   i. a Domain 1, comprising            -   (1) a sub-Domain (1A), which comprises a VL Domain of a                monoclonal antibody capable of binding to CD3 (VLCD3);                and            -   (2) a sub-Domain (1B), which comprises a VH Domain of a                monoclonal antibody capable of binding to HIV-1                (VHHIV-1), wherein the sub-Domains 1A and 1B are                separated from one another by a peptide linker (e.g. SEQ                ID NO:1);        -   ii. a Domain 2, wherein the Domain 2 is an E-coil Domain            (e.g. SEQ ID NO:7) or a K-coil Domain (e.g. SEQ ID NO:8),            wherein the Domain 2 is separated from the Domain 1 by a            peptide linker (SEQ ID NO:2); and    -   B. the second polypeptide chain comprises, in the N-terminal to        C-terminal direction:        -   i. a Domain 1, comprising            -   (1) a sub-Domain (1A), which comprises a VL Domain of a                monoclonal antibody capable of binding to HIV-1                (VLHIV-1); and            -   (2) a sub-Domain (1B), which comprises a VH Domain of a                monoclonal antibody capable of binding to CD3 (VHCD3),                wherein the sub-Domains 1A and 1B are separated from one                another by a peptide linker (e.g. SEQ ID NO:1); and        -   ii. a Domain 2, wherein the Domain 2 is a K-coil Domain            (e.g. SEQ ID NO:8) or an E-coil Domain (SEQ ID NO:7),            wherein the Domain 2 is separated from the Domain 1 by a            peptide linker (SEQ ID NO:2); and wherein the Domain 2 of            the first and the second polypeptide chains are not both            E-coil Domains or both K-coil Domains            and wherein:            (a) the VL Domain of the first polypeptide chain and the VH            Domain of the second polypeptide chain form an Antigen            Binding Domain capable of specifically binding to an epitope            of CD3; and            (b) the VL Domain of the second polypeptide chain and the VH            Domain of the first polypeptide chain form an Antigen            Binding Domain capable of specifically binding to HIV-1            envelope.

A bispecific molecule capable of specific binding to HIV-lenvelope andto an epitope of CD16, wherein the bispecific molecule comprises a firstpolypeptide chain and a second polypeptide chain, covalently bonded toone another, wherein:

-   -   A. the first polypeptide chain comprises, in the N-terminal to        C-terminal direction:        -   i. a Domain 1, comprising            -   (1) a sub-Domain (1A), which comprises a VL Domain of a                monoclonal antibody capable of binding to CD16 (VLCD16);                and            -   (2) a sub-Domain (1B), which comprises a VH Domain of a                monoclonal antibody capable of binding to HIV-1                (VHHIV-1), wherein the sub-Domains 1A and 1B are                separated from one another by a peptide linker (e.g. SEQ                ID NO:1);        -   ii. a Domain 2, wherein the Domain 2 is an E-coil Domain            (SEQ ID NO:7) or a K-coil Domain (e.g. SEQ ID NO:8), wherein            the Domain 2 is separated from the Domain 1 by a peptide            linker (SEQ ID NO:2); and    -   B. the second polypeptide chain comprises, in the N-terminal to        C-terminal direction:        -   i. a Domain 1, comprising            -   (1) a sub-Domain (1A), which comprises a VL Domain of a                monoclonal antibody capable of binding to HIV-1                (VLHIV-1); and            -   (2) a sub-Domain (1B), which comprises a VH Domain of a                monoclonal antibody capable of binding to CD16 (VHCD16),                wherein the sub-Domains 1A and 1B are separated from one                another by a peptide linker (e.g. SEQ ID NO:1); and        -   ii. a Domain 2, wherein the Domain 2 is a K-coil Domain e.g.            SEQ ID NO:8) or an E-coil Domain (e.g. SEQ ID NO:7), wherein            the Domain 2 is separated from the Domain 1 by a peptide            linker (SEQ ID NO:2); and wherein the Domain 2 of the first            and the second polypeptide chains are not both E-coil            Domains or both K-coil Domains            and wherein:            (a) the VL Domain of the first polypeptide chain and the VH            Domain of the second polypeptide chain form an Antigen            Binding Domain capable of specifically binding to an epitope            of CD16; and            (b) the VL Domain of the second polypeptide chain and the VH            Domain of the first polypeptide chain form an Antigen            Binding Domain capable of specifically binding to HIV-1            envelope.

In certain embodiments, the bispecific molecule binds to the HIV-1envelope like the HIV antibody from which it is derived. In certainembodiments, the bispecific molecule binds to the A32-HIV-1 envelopeepitope, i.e. the bispecific molecule binds to the HIV-1 envelope likethe A32 antibody, and CD3, or CD16. In certain embodiments, thebispecific molecule binds to the 7B2-HIV1 envelope epitope and CD3, orCD16. In certain embodiments, the bispecific molecule binds to theCH27-HIV-1 envelope epitope and CD3, or CD16. In certain embodiments,the bispecific molecule binds to the CH28-HIV-1 envelope epitope andCD3, or CD16. In certain embodiments, the bispecific molecule binds tothe CH44-HIV-1 envelope epitope and CD3, or CD16.

In certain embodiments, the bispecific molecule has the bindingspecificity of the A32 HIV-1-envelope antibody. In certain embodiments,the bispecific molecule has the binding specificity of the 7B2HIV-1-envelope antibody. The bispecific molecule has the bindingspecificity of the CH27 HIV-1-envelope antibody. The bispecific moleculehas the binding specificity of the CH28 HIV-1-envelope antibody. Incertain embodiments, the bispecific molecule has the binding specificityof the CH44 HIV-1-envelope antibody.

In certain embodiments a bispecific molecule of the invention comprises,consists essentially of or consists of sequences as described herein,e.g. Table 2 and Table 3)

In certain embodiments a bispecific molecule of the invention comprises,consists essentially of or consists of SEQ ID NO: 9 and 11; SEQ ID NO:13 and 15, SEQ ID NO: 17 and 19; SEQ ID NO; 21 and 23; SEQ ID NO: 25 and27; SEQ ID NO; 44 and 45 (See Table 2 and Table 3).

In certain aspects the invention provides compositions comprising any ofthe bispecific molecule described herein, or a combination thereof. Incertain embodiments, these compositions are formulated as pharmaceuticalcomposition for therapeutic use.

In certain aspects the invention is directed to nucleic acids whichencode the bispecific molecules of the invention. In certainembodiments, these nucleic acids are comprised in a vector, and areoperably linked to a promoter. In certain aspects the invention providescell lines, or isolated cells, which comprise nucleic acids for theexpression of the bispecific molecules of the invention.

In certain aspects, the invention provides compositions comprising thebispecific molecules of the invention or nucleic acids encoding the samefor use in methods of treating or preventing HIV infection. In someembodiments, these methods further comprise administering a LatencyActivating Reagent. Non-limiting examples of these include HDACinhibitors, e,g, vorinostat, romidepsin, panobinostat, disulfiram, JQ1,bryostatin, PMA, inonomycin, or any combination thereof. In someembodiments, this combination therapy targets the pool of latentlyinfected HIV cells.

In certain aspects, the invention provides methods treating orpreventing an HIV infection in a subject, the method comprisingadministering to the subject a composition comprising any one of thebispecific molecules of the invention, or a combination thereof in atherapeutically sufficient amount. In certain embodiments, the methodsfurther comprise administering a latency activating agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows potency of ADCC-mediating mAbs. The ADCC activity of the 5CHAVI mAbs against the 22 HIV-1 IMC is reported as maximum percentage ofspecific killing. Each dot represent the average activity of all thepositive results for each group of mAbs against the individual IMCs. Thelines represent the mean±standard deviation. The black line representthe cut-off for positive response.

FIG. 2 shows anti-HIV-1-DARTs-mediated cytotoxic activity. ActivatedCD4+ T cells from a HIV-1 seronegative donor were infected with HIV-1subtype B BaL, AE CM235, and C 1086.c IMC (top to bottom). The cellswere incubated with autologous resting CD8 T cells in the presence ofsix concentrations of the anti-HIV-1 (A32×CD3 ♦ and 7B2×CD3▪) andcontrol (4420×CD3⋅) DARTs for 6, 24, and 48 hours at an effector totarget cell ratio of 33:1. The results are reported as maximumpercentage of specific killing observed at each time point.

FIG. 3 shows dose dependence of anti-HIV-1 BaL DARTs-mediated cytotoxicactivity. Activated CD4+ T cells from a HIV-1 seronegative donor wereinfected with HIV-1 subtype B BaL. The cells were incubated withautologous resting CD8 T cells in the presence of six concentrations ofthe anti-HIV-1 (A32×CD3 ♦ and 7B2×CD3▪) and control (4420×CD3⋅) DARTsfor 48 hours at an effector to target cell ratio of 33, 11, and 3:1 (topto bottom). The results are reported as percentage of specific killing.

FIG. 4. shows DART concentration to reach 50% Specific Killing.Activated CD4+ T cells from a HIV-1 seronegative donor were infectedwith HIV-1 subtype B BaL, AE CM235, and C 1086.c IMC. The cells wereincubated with autologous resting CD8 T cells in the presence of sixconcentrations of the anti-HIV (A32×CD3, red; 7B2×CD3, blue) and control(4420×CD3, black) DARTs for 48 hours at an effector to target cell ratioof 33:1. Each bar represent the concentration required to detect 50%specific killing against each infected target population.

FIG. 5 shows the sequences of CH27, CH28 and CH44 HIV-1 antibodies. CDRsare indicated in the sequences (SEQ ID Nos: 57-74).

FIG. 6 shows the nucleotide sequences encoding VH and VL chains of A32antibody and amino acid sequences of VH and VL chains of A32 (SEQ ID Nos75-78 in order of appearance).

FIG. 7 shows nucleotide sequences encoding VH and VK chains of 7B2antibody and amino acid sequences of VH and VK chains of 7B2 (SEQ ID NO:79-82 in order of appearance).

FIG. 8A-C show the structures and domains of the bispecific molecules ofthe present invention. FIG. 8A illustrates the structure of a bispecificmolecule composed of two polypeptide chains. FIGS. 8B and 8C illustratethe structures of two versions of the first, second and thirdpolypeptide chains of a three chain bispecific molecule with an Fcdomain (Version 1, FIG. 8B; Version 2, FIG. 8C).

FIG. 9 shows various sequences: Linker 1 (SEQ ID NO; 1); Linker 2 (SEQID NO: 2); Heterodimer promoting domain and K-coil and E coil sequences(SEQ ID Nos: 3-6, land 8); Linker 3 (DKTHTCPPCP (SEQ ID No: 49); Linker4—SEQ ID NOS: 39, 40; CH2-CH3 fragments—SEQ ID Nos; 41-43; CH3 VHchain—SEQ ID NO: 51; CD3VL chain—SEQ ID NO: 52, CD16VH chain—SEQ ID NO53, CH16 VL chain—SEQ ID NO: 54; 7B2 VL—SEQ IDNO 55; 7b2 VH-SEQ ID NO56. SEQ ID Nos: 9-38, 44-48 show various bispecific antibodies (SeeTable 2).

FIGS. 10A-10C show HIV×CD3 DART structure. (FIGS. 10A-10B) These DARTmolecules contain an anti-HIV-1 binding arm (A32 or 7B2) combined withan anti-CD3 binding arm (hXR32). They are composed of two polypeptidechains: one with the VL of anti-CD3 linked to the VH of anti-HIV; thesecond with the VL of anti-HIV linked to the VH of anti-CD3. The carboxytermini of the chains have an interchain disulfide bond and pairedoppositely charged E-coil/K-coil dimerization domains. Control DARTshave one of the arms replaced by an irrelevant one derived from ananti-FITC antibody (4420) or from an anti-RSV antibody, palivizumab(RSV) sequence. (FIG. 10C) Schematic representation of HIV×CD3 DARTbinding to two distinct antigens simultaneously and redirecting thecytotoxic T cells (effectors) to lyse the Env-expressing, HIV-1 infectedcells (targets).

FIGS. 11A-11F show HIV×CD3 DART binding properties. FIGS. 11A-11C showantigen binding by ELISA. DART binding to human CD3 protein (FIG. 11A),to JR-FL gp140 protein (FIG. 11B) or simultaneously to both JR-FL gp140and human CD3 proteins (FIG. 11C). FIGS. 11D-11F show cell surfacebinding by FACS. DART binding to primary human T cells expressing CD3(FIG. 11D), to HEK293-D371 cells expressing HIV-1 Env, CM244, subtype AE(FIG. 11E) or to Jurkat 522-F/Y cells expressing CD3 and HIV-1 Env,HXBC2, subtype B (FIG. 11F). Data are reported as mean fluorescenceintensity (MFI). CD3 and Env expression characteristics of the cells arereported in parenthesis. A32 and 7B2 are targeting arms that recognizeHIV-1 gp120 and gp41, respectively; CD3 is the effector arm thatrecognizes CD3c; 4420 is an irrelevant, negative control arm.

FIGS. 12A-12H show HIV×CD3 DART redirected T-cell killing of Env⁺ targetcells. FIG. 12A shows DART concentration dependent killing of Env⁺Jurkat 522-F/Y cells in the presence of human T-cells at an E:T ratio of10:1 for 48 hours with cytolysis measured by LDH release assay; EC₅₀values were 230 and 160 pg/mL for A32×CD3 and 7B2×CD3, respectively. Thecontrol DARTs (A32×4420, 7B2×4420, 4420×CD3) were inactive. FIG. 12Bshows lack of DART mediated killing of Env⁺ Jurkat 522-F/Y cells in theabsence of effector T-cells with cytolysis measured by LDH releaseassay. FIG. 12C shows lack of DART redirected T-cell killing of Env⁻Jurkat ΔKS cells at an E:T ratio of 10:1 for 48 hours with cytolysismeasured by LDH release assay. FIG. 12D shows DART concentrationdependent killing of Env⁺ Jurkat 522-F/Y GF cells in the presence ofhuman T-cells at an E:T ratio of 10:1 for 48 hours with cytolysismeasured by LUM assay; EC₅₀ values were 172 and 147 pg/mL for A32×CD3and 7B2×CD3, respectively. FIGS. 12E-12G show 7B2×CD3 DART concentrationdependent redirected T cell killing of Env⁺ Jurkat 522-F/Y GF cells atdifferent E:T ratios (10:1, 5:1, 1:1) and incubation times (24, 48, 72hours) with cytolysis measured by LUM assay. FIG. 12H shows time courseof maximal cytolytic activity with 7B2×CD3 at different E:T ratios (datafrom FIGS. 12E-12G).

FIGS. 13A-13F show HIV×CD3 DARTs redirect T-cell cytotoxicity againstCD4+ cells infected with HIV-1 IMCs of different subtypes. FIGS. 13A-13Cshow DART concentration dependence. Activated CD4+ T cells from a HIV-1seronegative donor were infected with HIV-1 subtype B BaL (FIG. 13A),subtype AE CM235 (FIG. 13B) or subtype C 1086.0 (FIG. 13C) IMC andincubated for 48 hours with A32×CD3 (red circles), 7B2×CD3 (bluesquares) or 4420×CD3 (black diamonds) in the presence of autologousresting CD8⁺ T cells at an E:T ratio of 33:1 (filled symbols) or in theabsence of effector cells (E:T ratio of 0:1) (open symbols). The dataare reported as percentage of specific lysis (% SL). DART concentrationsranged from 0.001 to 1000 ng/mL. FIGS. 13D-13F show time course. Thedata represent the maximal % SL observed at 6, 24, and 48 hours for eachDART against CD4+ T cells infected with HIV-1 subtype B BaL (FIG. 13D),subtype AE CM235 (FIG. 13E) or subtype C 1086.0 (FIG. 13F) IMC andincubated with autologous resting CD8⁺ T cells at an E:T ratio of 33:1.

FIGS. 14A-14H show HIV×CD3 DARTs induce specific degranulation of CD8+T-cell. FIGS. 14A-14D show schematic of gating strategy to identifyLive/CD3+CD8+CD107+ T cells after their incubation with HIV-1 BaLinfected target cells in presence of DARTs for 6 hours. (FIGS. 14E-14G)Dot plots represent the percentage of Live/CD3⁺ CD8⁺ CD107⁺ cellsobserved in presence of 1 ng/mL of 4420×CD3 (FIG. 14E), 7B2×CD3 (FIG.14F) or A32×CD3 (FIG. 14G). FIG. 14H shows frequency of the CD3⁺ CD4⁻CD8⁺ CD107⁺ T cells observed in each of the five HIV-1 seronegativehealthy donors after 6 hours of incubation with the autologous infectedCD4⁺ T cells using the E:T ratio of 33:1. Each symbol represents theaverage of duplicate stimulations performed for each donor. The linesrepresent the mean±standard deviation. * indicates p<0.05 afterDunnett's test for multiple comparisons.

FIGS. 15A-15C show viral clearance assay to assess HIV×CD3 DARTredirected CD8+ T cell killing of autologous JR-CSF-infected CD4+ Tcells from healthy HIV seronegative donors. Activated CD4⁺ T cells fromHIV seronegative donors were infected with HIV-1 clone JR-CSF and thenincubated with autologous resting CD8⁺ T effector cells at an E:T ratioof 1:1 in the absence (No DART) or presence of HIV×CD3 or control DARTsat a concentration of 100 ng/mL for 7 days. Results are shown for twohealthy donors (FIGS. 15A-15B), as well as for healthy donor 2 in thepresence of integrase and non-nucleoside reverse transcriptaseinhibitors during the co-culture period to inhibit virus replication(FIG. 15C). Each bar represents the absolute p24 concentration detectedin culture supernatants. Error bars represent standard error mean (SEM)of n=3. * indicates p<0.05 with Dunnett's test for multiple comparisons.

FIGS. 16A-16H show viral clearance assay detects HIV×CD3 DART redirectedCD8+ T-cell clearance of JR-CSF or autologous reservoir (AR)virus-infected CD4+ cells using lymphocytes from HIV-infected ARTsuppressed patients. CD4⁺ depleted T cells from HIV-infected ARTsuppressed patients were activated with PHA and infected with HIV-1subtype B clone JR-CSF (FIGS. 16A-16C) or autologous reservoir (AR)virus isolates (FIGS. 16D-16F) and then incubated without (FIGS. 16A,16D) or with autologous CD8⁺ T effector cells at E:T ratios of 1:10(FIGS. 16B, 16E) or 1:1 (FIGS. 16C, 16F) in the absence (No DART) orpresence of HIV×CD3 (A32×CD3, 7B2×CD3) or control (7B2×4420, 4420×CD3)DARTs at a concentration of 100 ng/mL for 7 days. ‘Combo’ indicates a1:1 cocktail of 7B2×CD3 and A32×CD3 at a total concentration of 100ng/mL. Each bar represents the log fold reduction of p24 detected inculture supernatants, calculated as the log (p24 of infected targetcells only control divided by p24 of the test condition). FIG. 16G showsschematic of gating strategy to identify Live/CD3+CD4+CD107+ Effector(TFL4-) T cells after their incubation with HIV-1 JR-CSF infected targetcells in presence of DARTs for 6 hours. FIG. 16H shows the % oflive/effector cells (TFL4 negative)/CD3+/CD4+/107a+ cells following a 6hour incubation with the indicated DARTs and JR-CSF infected targets inn=4 patients. Error bars represent SEM of n=8 (FIGS. 16A-16C, except forcombo n=5 and 7B2×4420 n=6), n=5 (FIGS. 16D-16F), and n=4 (FIGS.16G-16H). * indicates p<0.05 with Dunnett's test for multiplecomparisons.

FIGS. 17A-17B show latency clearance assay to assess HIV×CD3 DARTredirected CD8⁺ T-cell activity. Resting CD4⁺ T cells from HIV-infected,ART suppressed patients were incubated with PHA (FIG. 17A) or vorinostat(FIG. 17B), plated in 12-36 replicate wells depending on the size of thepatient's latent reservoir, and co-cultured with autologous CD8⁺ T cellsat an E:T ratio of 1:10 in the absence or presence of HIV×CD3 or controlDARTs at 100 ng/mL for 24 hours (or up to 96 hours where indicated),after which DARTs were washed off and CD8-depleted PBMCs from aseronegative donor were added to amplify residual virus. Wells wereassessed for the presence or absence of p24 by ELISA at day 15. ‘Combo’indicates a 1:1 cocktail of 7B2×CD3 and A32×CD3 at a total concentrationof 100 ng/mL. Results are shown as % viral recovery (# of positivewells/total number plated), normalized to a control in which no CD8⁺ Tcells are added. Dashed lines indicate undetectable viral recovery. NTindicated the conditions that were not tested due to low cellavailability according to the table shown in FIG. 21.

FIG. 18 shows a list of IMC by HIV-1 Subtypes and Neutralization Tier.

FIG. 19 shows Equilibrium Dissociation Constants (K_(D)) for Binding ofA32×CD3 and 7B2×CD3 to Recombinant Env and CD3 Protein.

FIG. 20 shows Clinical Characteristics.

FIG. 21 shows that DARTs redirect patient T cells against JR-CSFinfected autologous target cells and absolute p24 concentration.

FIG. 22 shows Absolute # of Positive Wells in Latency Clearance Assaywith DARTs.

FIG. 23 shows potency and breadth of ADCC-mediating mAbs. The ADCCactivities of the A32 (anti-gp120 C1/C2) mAb (♦) and 7B2 (anti-gp41cluster I) mAb (▪) are reported as maximum percentage of specific lysis(% SL) against each of the 22 HIV-1 IMC. Each dot represents one HIV-1IMC. The results obtained with plasma from one HIV-1 seropositive(positive control; pos ctrl) and one seronegative (negative control; negctrl) donor are also reported. The lines represent the mean±standarddeviation. The black line represents the cut-off for positive response.

FIG. 24 shows conservation of HIV-1 Env residues known to influence thebinding of 7B2 and A32 mAbs. A linear 7-residue sequence in gp41 (gp160positions 598-604; immunodominant cluster I) is reported to contain thebinding site for 7B2 mAb (28, 29). Discontinuous residues in gp120 C1-C4known to influence A32 mAb binding (based on point mutagenesis studies)occur at positions 52, 53, 66, 69, 83, 86, 96, 100, 103, 107, 112, 215,217, 252, 256, 262, 427 and 479 (37, 39, 68). The conservation of theseresidues in the Los Alamos National Laboratory (LANL) HIV1 Env Aminoacid Filtered web alignment, a database consisting of 4556 HIV-1 Envsequences with representation of all subtypes, was assessed byQuickAlign analysis(http://www.hiv.lanl.gov/content/sequence/QUICK_ALIGNv2/QuickAlign.html).The height of the residue at each position of Env is proportional to itsfrequency of distribution among the HIV-1 isolates. Residues are coloredaccording to hydrophobicity: black, hydrophilic; green, neutral; blue,hydrophobic. Based on a crystal structure of a CD4-stabilized gp120 corecomplexed with a Fab fragment of N5-i5 (an A32-like mAb), residues at52, 53, 69, 103, 107 and 217 (located in C1-C2) may be direct epitopecontacts (27).

FIG. 25 shows cell surface Env binding of A32×4420 and 7B2×4420 controlDARTs. DART binding to HEK293-D371 cells expressing HIV-1 Env, CM244,subtype AE was measured and data are reported as mean fluorescenceintensity (MFI). A32 and 7B2 are targeting arms that recognize HIV-1gp120 and gp41, respectively; 4420 is an irrelevant, negative controlarm.

FIGS. 26A-26D show HIV×CD3 DART-mediated T-cell activation depends onco-engagement with target cells. Unstimulated CD4⁺ or CD8⁺ T-cells fromhealthy seronegative donors were incubated with (FIGS. 26A, 26C) andwithout (FIGS. 26B, 26D) Env expressing Jurkat-522 F/Y cell line in theabsence or presence of control (RSV×CD3) or HIV×CD3 (A32×CD3, 7B2×CD3)DARTs at 40, 0.32, and 0 ng/mL for 48 hours. CD8⁺(FIGS. 26A-26B) andCD4⁺ (FIGS. 26C-26D) T cell activation was assessed by staining withCD25 Ab cells. The data are reported as frequency (%) of activated(CD25⁺) T cells. Each bar represent the average of results obtained from2 different donors.

FIG. 27 shows HIV DARTs bind specifically to HIV-1 IMC infected CD4⁺ Tcells. Activated CD4⁺ T cells obtained from healthy HIV-1 seronegativedonors were infected for 48 hours with HIV-1 IMCs representing the HIV-1subtype B BaL, AE CM235, and C 1086.0 as reported in the methodssection. Non-infected CD4⁺ T cells (mock) were utilized as negativecontrol. The cells were stained using the 7B2×4420 and A32×4420 DARTwhere the CD3 arm was substituted with the irrelevant 4420 protein toavoid binding to the CD3 receptor. After incubation with the DART, thecells were stained with the secondary anti-EK-IgG2a-biotinylated complexto reveal binding of the DARTs. The staining with 7B2 and A32 mAbs,utilizing an indirect staining technique with the secondary mouseanti-human-IgG mAb, was performed as control. The secondaryfluoresceinated anti-human IgG Abs and the Palivizumab mAb were utilizedas negative controls. The frequency of infected cells was determined byintracellular staining using the anti-p24 mAb as reported in the methodsection. Each bar represents CD4⁺ T cells infected with the IMCs andcontrols as indicated above the graph. The results are reported asfrequency (%) of viable infected (p24⁺) CD4⁺ T cells that were stainedby each of the DARTs, mAbs, and controls as listed on the x-axis.

FIGS. 28A-28D show lack of HIV×CD3 DART effects on T cell viability oractivation status in the absence of added target cells using PBMC fromHIV-1 infected donors. Unstimulated CD4⁺ or CD8⁺ T-cells fromHIV-infected, ART suppressed were incubated in the absence or presenceof control (4420×CD3, 7B2×4420, A32×4420) or active (A32×CD3, 7B2×CD3)DARTs at 100 ng/mL for 7 days. (FIGS. 28A-28B) T cell viability wasassessed by staining cells for Annexin V/7-AAD. Viable cells wereidentified as those that were Annein V and 7-AAD negative. (FIGS.28C-28D) T cell activation was assessed by staining cells for HLA-DR andCD25 expression. Data points for both analyses are from n=3 patientsperformed on 3 independent occasions. Error bars represent standarderror mean.

DETAILED DESCRIPTION

Highly active anti-retroviral therapy (HAART) alone or in combinationwith latency reversing agents fails to reduce the pool of latentlyinfected cells. This is due to limited ability of the CD8+ T cells toeliminate HIV-1 latently infected cells. Dual Affinity Re-Targetingproteins (DARTs) are bispecific, antibody-based molecules that can bindtwo distinct antigens simultaneously. HIV-1 DARTs contain an HIV-1binding arm combined with an effector cell binding arm, and are designedto redirect cytotoxic CD3+ T cells to engage and kill HIV-infectedcells. A panel of monoclonal antibodies (mAbs) was studied to determinetheir magnitude and breadth of mediating ADCC against 22 differentisolates. The goals were to: 1) identify mAbs that could be used as theHIV-1 binding arms of DARTs; 2) test the resulting DARTs for theirability to mediate killing of HIV-1 infected cells. Provided herein aredata related to the potency of the different groups of ADCC-mediatingmAbs and the resulting DARTs against HIV-1 Infectious Molecular Clones(IMC)-infected target cells.

Antibodies and Other Binding Molecules

Antibodies

The invention provides polyclonal or monoclonal antibodies, variants,fusion proteins comprising an antibody portion with an antigenrecognition site of the required specificity, humanized antibodies, andchimeric antibodies, and any other modified configuration of theimmunoglobulin molecule that comprises an antigen recognition site ofthe required specificity. Throughout this application, the numbering ofamino acid residues of the light and heavy chains of antibodies isaccording to the EU index as in Kabat et al. (1992) SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, National Institutes of HealthPublication No. 91-3242. In some embodiments, antigen-binding fragmentof an antibody is a portion of an antibody that possesses an at leastone antigen recognition site. Fragments include for example but notlimited to Fab, Fab′, F(ab′)₂ Fv), and single chain (scFv).

Monoclonal antibodies are known in the art. In certain embodiments,monoclonal antibody encompasses not only intact monoclonal antibodiesand full-length monoclonal antibodies, but also fragments thereof (suchas Fab, Fab′, F(ab′)₂ Fv), single chain (scFv), mutants thereof, fusionproteins comprising an antibody portion, humanized monoclonalantibodies, chimeric monoclonal antibodies, and any other modifiedconfiguration of the immunoglobulin molecule that comprises an antigenrecognition site of the required specificity and the ability to bind toan antigen. Monoclonal antibodies are not limited as regards to thesource of the antibody or the manner in which it is made (e.g., byhybridoma, phage selection, recombinant expression, transgenic animals,etc.).

Methods of making monoclonal antibodies are known in the art. In certainembodiments, the antibodies are produced recombinantly by any meansknown in the art. In one embodiment, such an antibody is sequenced andthe polynucleotide sequence is then cloned into a vector for expressionor propagation. The sequence encoding the antibody of interest may bemaintained in a vector in a host cell and the host cell can then beexpanded and frozen for future use. The polynucleotide sequence of suchantibodies may be used for genetic manipulation to generate thebi-specific molecules of the invention as well as a chimeric antibody, ahumanized antibody, or a caninized antibody, to improve the affinity, orother characteristics of the antibody. The general principle inhumanizing an antibody involves retaining the basic sequence of theantigen-binding portion of the antibody, while swapping the non-humanremainder of the antibody with human antibody sequences. There are fourgeneral steps to humanize a monoclonal antibody. These are: (1)determining the nucleotide and predicted amino acid sequence of thestarting antibody light and heavy variable Domains (2) designing thehumanized antibody or caninized antibody, i.e., deciding which antibodyframework region to use during the humanizing or canonizing process (3)the actual humanizing or caninizing methodologies/techniques and (4) thetransfection and expression of the humanized antibody. See, for example,U.S. Pat. Nos. 4,816,567; 5,807,715; 5,866,692; and 6,331,415.

Bi-Specific Antibodies, Multi-Specific Diabodies and DART™ Diabodies

The provision of non-mono-specific “diabodies” provides a significantadvantage over antibodies: the capacity to co-ligate and co-localizecells that express different epitopes. Bivalent diabodies thus havewide-ranging applications including therapy and immunodiagnosis.Bi-valency allows for great flexibility in the design and engineering ofthe diabody in various applications, providing enhanced avidity tomultimeric antigens, the cross-linking of differing antigens, anddirected targeting to specific cell types relying on the presence ofboth target antigens. Due to their increased valency, low dissociationrates and rapid clearance from the circulation (for diabodies of smallsize, at or below 50 kDa), diabody molecules known in the art have alsoshown particular use in the field of tumor imaging (Fitzgerald et al.(1997) “Improved Tumour Targeting By Disulphide Stabilized DiabodiesExpressed In Pichia pastoris,” Protein Eng. 10:1221). Of particularimportance is the co-ligating of differing cells, for example, thecross-linking of cytotoxic T cells to tumor cells (Staerz et al. (1985)“Hybrid Antibodies Can Target Sites For Attack By T Cells,” Nature314:628-631, and Holliger et al. (1996) “Specific Killing Of LymphomaCells By Cytotoxic T-Cells Mediated By A Bispecific Diabody,” ProteinEng. 9:299-305).

Diabody epitope binding domains may also be directed to a surfacedeterminant of a B cell, such as CD19, CD20, CD22, CD30, CD37, CD40, andCD74 (Moore, P. A. et al. (2011) “Application Of Dual AffinityRetargeting Molecules To Achieve Optimal Redirected T-Cell Killing OfB-Cell Lymphoma,” Blood 117(17):4542-4551; Cheson, B. D. et al. (2008)“Monoclonal Antibody Therapy For B-Cell Non Hodgkin's Lymphoma,” N.Engl. J. Med. 359(6):613-626; Castillo, J. et al. (2008) “Newermonoclonal antibodies for hematological malignancies,” Exp. Hematol.36(7):755-768. In many studies, diabody binding to effector celldeterminants, e.g., Fcγ receptors (FcγR), was also found to activate theeffector cell (Holliger et al. (1996) “Specific Killing Of LymphomaCells By Cytotoxic T-Cells Mediated By A Bispecific Diabody,” ProteinEng. 9:299-305; Holliger et al. (1999) “Carcinoembryonic Antigen(CEA)-Specific T-Cell Activation In Colon Carcinoma Induced ByAnti-CD3×Anti-CEA Bispecific Diabodies And B7×Anti-CEA Bispecific FusionProteins,” Cancer Res. 59:2909-2916; WO 2006/113665; WO 2008/157379; WO2010/080538; WO 2012/018687; WO 2012/162068). Normally, effector cellactivation is triggered by the binding of an antigen bound antibody toan effector cell via Fc-FcγR interaction; thus, in this regard, diabodymolecules may exhibit Ig-like functionality independent of whether theycomprise an Fc Domain (e.g., as assayed in any effector function assayknown in the art or exemplified herein (e.g., ADCC assay)). Bycross-linking tumor and effector cells, the diabody not only brings theeffector cell within the proximity of the tumor cells but leads toeffective tumor killing (see e.g., Cao et al. (2003) “BispecificAntibody Conjugates In Therapeutics,” Adv. Drug. Deliv. Rev.55:171-197).

The formation of such non-mono-specific diabodies requires thesuccessful assembly of two or more distinct and different polypeptides(i.e., such formation requires that the diabodies be formed through theheterodimerization of different polypeptide chain species). This fact isin contrast to mono-specific diabodies, which are formed through thehomodimerization of identical polypeptide chains. Because at least twodissimilar polypeptides (i.e., two polypeptide species) must be providedin order to form a non-mono-specific diabody, and becausehomodimerization of such polypeptides leads to inactive molecules(Takemura, S. et al. (2000) “Construction Of A Diabody (SmallRecombinant Bispecific Antibody) Using A Refolding System,” Protein Eng.13(8):583-588), the production of such polypeptides must be accomplishedin such a way as to prevent covalent bonding between polypeptides of thesame species (i.e., so as to prevent homodimerization) (Takemura, S. etal. (2000) “Construction Of A Diabody (Small Recombinant BispecificAntibody) Using A Refolding System,” Protein Eng. 13(8):583-588). Theart has therefore taught the non-covalent association of suchpolypeptides (see, e.g., Olafsen et al. (2004) “CovalentDisulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation AndRadiolabeling For Tumor Targeting Applications,” Prot. Engr. Des. Sel.17:21-27; Asano et al. (2004) “A Diabody For Cancer Immunotherapy AndIts Functional Enhancement By Fusion Of Human Fc Domain,” Abstract3P-683, J. Biochem. 76(8):992; Takemura, S. et al. (2000) “ConstructionOf A Diabody (Small Recombinant Bispecific Antibody) Using A RefoldingSystem,” Protein Eng. 13(8):583-588; Lu, D. et al. (2005) “A Fully HumanRecombinant IgG-Like Bispecific Antibody To Both The Epidermal GrowthFactor Receptor And The Insulin-Like Growth Factor Receptor For EnhancedAntitumor Activity,” J. Biol. Chem. 280(20):19665-19672).

The art has recognized that bi-specific diabodies composed ofnon-covalently associated polypeptides are unstable and readilydissociate into non-functional monomers (see, e.g., Lu, D. et al. (2005)“A Fully Human Recombinant IgG-Like Bispecific Antibody To Both TheEpidermal Growth Factor Receptor And The Insulin-Like Growth FactorReceptor For Enhanced Antitumor Activity,” J. Biol. Chem.280(20):19665-19672).

In the face of this challenge, the invention provides stable, covalentlybonded heterodimeric non-mono-specific diabodies, termed DARTs™ (see,e.g., United States Patent Publications No. 2014-0099318; 2013-0295121;2010-0174053 and 2009-0060910; European Patent Publication No. EP2714079; EP 2601216; EP 2376109; EP 2158221 and PCT Publications No. WO2015/026894; WO2015/026892; WO 2015/021089; WO 2014/159940; WO2012/162068; WO 2012/018687; WO 2010/080538; Moore, P. A. et al. (2011)“Application Of Dual Affinity Retargeting Molecules To Achieve OptimalRedirected T-Cell Killing Of B-Cell Lymphoma,” Blood 117(17):4542-4551;Veri, M. C. et al. (2010) “Therapeutic Control Of B Cell Activation ViaRecruitment Of Fcgamma Receptor IIb (CD32B) Inhibitory Function With ANovel Bispecific Antibody Scaffold,” Arthritis Rheum. 62(7):1933-1943;Johnson, S. et al. (2010) “Effector Cell Recruitment With Novel Fv-BasedDual-Affinity Re-Targeting Protein Leads To Potent Tumor Cytolysis Andin vivo B-Cell Depletion,” J. Mol. Biol. 399(3):436-449), the contentsof which publications are herein incorporated by reference in theirentirety. Such diabodies comprise two or more covalently complexedpolypeptides and involve engineering one or more cysteine residues intoeach of the employed polypeptide species. For example, the addition of acysteine residue to the c-terminus of such constructs has been shown toallow disulfide bonding between the polypeptide chains, stabilizing theresulting heterodimer without interfering with the bindingcharacteristics of the bivalent molecule.

In some embodiments, each of the two polypeptides of the DART™ comprisesthree Domains (FIG. 8A). The first polypeptide comprises: (i) a Domainthat comprises a binding region of a light chain variable Domain of thea first immunoglobulin (VL1), (ii) a second Domain that comprises abinding region of a heavy chain variable Domain of a secondimmunoglobulin (VH2), and (iii) a third Domain that serves to promoteheterodimerization with the second polypeptide and to covalently bondthe first polypeptide to the second polypeptide of the diabody. Thesecond polypeptide contains a complementary first Domain (a VL2 Domain),a complementary second Domain (a VH1 Domain) and a third Domain thatcomplexes with the third Domain of the first polypeptide chain in orderto promote heterodimerization and covalent bonding with the firstpolypeptide chain. Such molecules are stable, potent and have theability to simultaneously bind two or more antigens. They are able topromote redirected T cell (CD3) or NK (CD16) cell mediated killing ofcells expressing target antigens.

In certain aspects, the present invention is directed to HIV-1×CD3 andHIV-1×CD16 bi-specific monovalent diabodies that are capable ofsimultaneous binding to HIV-1 and CD3 or HIV-1 and CD16, and to the usesof such molecules in the treatment of HIV-1 infection.

In certain embodiments, the HIV-1×CD3 and HIV-1×CD16 bi-specificmonovalent diabodies of the present invention are composed of twopolypeptide chains which associate with one another to form one bindingsite specific for an epitope of HIV-1 and one binding site specific foran epitope of CD3 or CD16 (see, FIG. 8), so as to be capable ofsimultaneously binding to HIV-1 and to CD3 or CD16. Thus, such diabodiesbind to a “first antigen,” which may be either CD3 or HIV-1, and a“second antigen,” which is HIV-1 when the first epitope is CD3, and isCD3 when the first epitope is HIV-1. Alternatively, such diabodies bindto a “first antigen,” which may be either CD16 or HIV-1, and a “secondantigen,” which is HIV-1 when the first epitope is CD16, and is CD16when the first epitope is HIV-1.

In certain embodiments as shown in FIG. 8, the first of such twopolypeptide chains will contain, in the N-terminal to C-terminaldirection, an N-terminus, the Antigen-Binding Domain of a Light ChainVariable Domain (VL) of a “first” antigen (either CD3 or HIV-1envelope), the Antigen-Binding Domain of a Heavy Chain Variable Domain(VH) of a second antigen (HIV-1, if the first antigen was CD3; CD3, ifthe first antigen was HIV-1), a Heterodimerization-Promoting Domain, anda C-terminus. An intervening linker peptide (Linker 1) separates theAntigen-Binding Domain of the Light Chain Variable Domain from theAntigen-Binding Domain of the Heavy Chain Variable Domain. In certainembodiments the Antigen-Binding Domain of the Heavy Chain VariableDomain is linked to the Heterodimerization-Promoting Domain by anintervening linker peptide (Linker 2). In certain embodiments the firstof the two polypeptide chains will thus contain, in the N-terminal toC-terminal direction: VL_(First Antigen)-Linker1-VH_(Second Antigen)-Linker 2-Heterodimerization-Promoting Domain.

In certain embodiments, the second of such two polypeptide chains willcontain, in the N-terminal to C-terminal direction, an N-terminus, theAntigen-Binding Domain of a Light Chain Variable Domain (VL) of thesecond antigen, the Antigen-Binding Domain of a Heavy Chain VariableDomain (VH) of the first antigen, a Heterodimerization-Promoting Domainand a C-terminus. An intervening linker peptide (Linker 1) separates theAntigen-Binding Domain of the Light Chain Variable Domain from theAntigen-Binding Domain of the Heavy Chain Variable Domain. In certainembodiments, the Antigen-Binding Domain of the Heavy Chain VariableDomain is linked to the Heterodimerization-Promoting Domain by anintervening linker peptide (Linker 2). In certain embodiments the secondof the two polypeptide chains will thus contain, in the N-terminal toC-terminal direction: VL_(Second Antigen)-Linker1-VH_(First Antigen)-Linker 2-Heterodimerization-Promoting Domain.

The Antigen-Binding Domain of the Light Chain Variable Domain of thefirst polypeptide chain interacts with the Antigen-Binding Domain of theHeavy Chain Variable Domain of the second polypeptide chain in order toform a functional antigen-binding site that is specific for the firstantigen (i.e., either HIV-1 envelope or CD3/CD16). Likewise, theAntigen-Binding Domain of the Light Chain Variable Domain of the secondpolypeptide chain interacts with the Antigen-Binding Domain of the HeavyChain Variable Domain of the first polypeptide chain in order to form asecond functional antigen-binding site that is specific for the secondantigen (i.e., either CD3/CD16 or HIV-1 envelope, depending upon theidentity of the first antigen). Thus, the selection of theAntigen-Binding Domain of the Light Chain Variable Domain and theAntigen-Binding Domain of the Heavy Chain Variable Domain of the firstand second polypeptide chains are coordinated, such that the twopolypeptide chains collectively comprise Antigen-Binding Domains oflight and Heavy Chain Variable Domains capable of binding to theintended targets, in certain embodiments e.g. HIV-1 envelope and CD3, orCD16.

In certain embodiments the length of Linker 1, which separates such VLand VH domains of a polypeptide chain is selected to substantially orcompletely prevent such VL and VH domains from binding to one another.Thus the VL and VH domains of the first polypeptide chain aresubstantially or completely incapable of binding to one another.Likewise, the VL and VH domains of the second polypeptide chain aresubstantially or completely incapable of binding to one another. Incertain embodiments this is due to the linker which separates the VH andVL domains. In certain embodiments, the linker is 5, 6, 7, 8, 9, 10, 11,12, 13, 14, but no more than 15 amino acids. In certain embodiments anintervening spacer peptide (Linker 1) has the sequence (SEQ ID NO:1):GGGSGGGG.

Linker 2 separates the VH Domain of a polypeptide chain from theHeterodimer-Promoting Domain of that polypeptide chain. Any of a varietyof linkers can be used for the purpose of Linker 2. In certainembodiments a sequence for such Linker 2 has the amino acid sequence:GGCGGG (SEQ ID NO:2), which has a cysteine residue that may be used tocovalently bond the first and second polypeptide chains to one anothervia a disulfide bond.

The formation of heterodimers of the first and second polypeptide chainscan be driven by the inclusion of Heterodimerization-Promoting Domains.Such domains include GVEPKSC (SEQ ID NO:3) or VEPKSC (SEQ ID NO:4) onone polypeptide chain and GFNRGEC (SEQ ID NO:5) or FNRGEC (SEQ ID NO:6)on the other polypeptide chain (See US2007/0004909 herein incorporatedby reference in its entirety).

In certain embodiments, the Heterodimerization-Promoting Domains of thepresent invention are formed from one, two, three or four tandemlyrepeated coil domains of opposing charge that comprise a sequence of atleast six, at least seven or at least eight charged amino acid residues(Apostolovic, B. et al. (2008) “pH-Sensitivity of the E3/K3Heterodimeric Coiled Coil,” Biomacromolecules 9:3173-3180; Arndt, K. M.et al. (2001) “Helix-stabilized Fv (hsFv) Antibody Fragments:Substituting the Constant Domains of a Fab Fragment for a HeterodimericCoiled-coil Domain,” J. Molec. Biol. 312:221-228; Arndt, K. M. et al.(2002) “Comparison of In Vivo Selection and Rational Design ofHeterodimeric Coiled Coils,” Structure 10:1235-1248; Boucher, C. et al.(2010) “Protein Detection By Western Blot Via Coiled—Coil Interactions,”Analytical Biochemistry 399:138-140; Cachia, P. J. et al. (2004)“Synthetic Peptide Vaccine Development: Measurement Of PolyclonalAntibody Affinity And Cross-Reactivity Using A New Peptide Capture AndRelease System For Surface Plasmon Resonance Spectroscopy,” J. Mol.Recognit. 17:540-557; De Crescenzo, G. D. et al. (2003) “Real-TimeMonitoring of the Interactions of Two-Stranded de novo DesignedCoiled-Coils: Effect of Chain Length on the Kinetic and ThermodynamicConstants of Binding,” Biochemistry 42:1754-1763; Fernandez-Rodriquez,J. et al. (2012) “Induced Heterodimerization And Purification Of TwoTarget Proteins By A Synthetic Coiled-Coil Tag,” Protein Science21:511-519; Ghosh, T. S. et al. (2009) “End-To-End And End-To-MiddleInterhelical Interactions: New Classes Of Interacting Helix Pairs InProtein Structures,” Acta Crystallographica D65:1032-1041; Grigoryan, G.et al. (2008) “Structural Specificity In Coiled-Coil Interactions,”Curr. Opin. Struc. Biol. 18:477-483; Litowski, J. R. et al. (2002)“Designing Heterodimeric Two-Stranded a-Helical Coiled-Coils: TheEffects Of Hydrophobicity And α-Helical Propensity On Protein Folding,Stability, And Specificity,” J. Biol. Chem. 277:37272-37279;Steinkruger, J. D. et al. (2012) “The d′-d-d′ Vertical Triad is LessDiscriminating Than the a′-a-a′ Vertical Triad in the AntiparallelCoiled-coil Dimer Motif,” J. Amer. Chem. Soc. 134(5):2626-2633;Straussman, R. et al. (2007) “Kinking the Coiled Coil—Negatively ChargedResidues at the Coiled-coil Interface,” J. Molec. Biol. 366:1232-1242;Tripet, B. et al. (2002) “Kinetic Analysis of the Interactions betweenTroponin C and the C-terminal Troponin I Regulatory Region andValidation of a New Peptide Delivery/Capture System used for SurfacePlasmon Resonance,” J. Molec. Biol. 323:345-362; Woolfson, D. N. (2005)“The Design Of Coiled—Coil Structures And Assemblies,” Adv. Prot. Chem.70:79-112; Zeng, Y. et al. (2008) “A Ligand-Pseudoreceptor System BasedOn de novo Designed Peptides For The Generation Of Adenoviral VectorsWith Altered Tropism,” J. Gene Med. 10:355-367).

Such repeated coil domains may be exact repeats or may havesubstitutions. For example, the Heterodimerization-Promoting Domain ofthe first polypeptide chain may comprise a sequence of eight negativelycharged amino acid residues and the Heterodimerization-Promoting Domainof the second polypeptide chain may comprise a sequence of eightnegatively charged amino acid residues. It is immaterial which coil isprovided to the first or second polypeptide chains, provided that a coilof opposite charge is used for the other polypeptide chain.

In certain embodiments an HIV-1×CD3 bi-specific monovalent diabody ofthe present invention has a first polypeptide chain having a negativelycharged coil. The positively charged amino acid may be lysine, arginine,histidine, etc. and/or the negatively charged amino acid may be glutamicacid, aspartic acid, etc. In certain embodiments the positively chargedamino acid is lysine and/or the negatively charged amino acid isglutamic acid. It is possible for only a singleHeterodimerization-Promoting Domain to be employed (since such domainwill inhibit homodimerization and thereby promote heterodimerization).In certain embodiments both the first and second polypeptide chains ofthe diabodies of the present invention to containHeterodimerization-Promoting Domains.

In certain embodiments, one of the Heterodimerization-Promoting Domainswill comprise four tandem “E-coil” helical domains (SEQ ID NO:7:EVAALEK-EVAALEK-EVAALEK-EVAALEK), whose glutamate residues will form anegative charge at pH 7, while the other of theHeterodimerization-Promoting Domains will comprise four tandem “K-coil”domains (SEQ ID NO:8: KVAALKE-KVAALKE-KVAALKE-KVAALKE), whose lysineresidues will form a positive charge at pH 7. The presence of suchcharged domains promotes association between the first and secondpolypeptides, and thus fosters heterodimerization. In some embodiments,the number of K coil and E coil domains can vary and a skilled artisancan readily determine whether a different number of K-coil or E-coildomain lead to heterodimerization.

In certain embodiments, the HIV-1×CD3 or HIV-1×CD16 bi-specificmonovalent diabodies of the present invention are engineered so thattheir first and second polypeptide chains covalently bond to one anothervia one or more cysteine residues positioned along their length. Suchcysteine residues may be introduced into the intervening linker thatseparates the VL and VH domains of the polypeptides. Alternatively,Linker 2 may contain a cysteine residue.

The invention also includes variants of the antibodies (and fragments)disclosed herein, including variants that retain the ability to bind torecombinant Env protein, the ability to bind to the surface ofvirus-infected cells and/or ADCC-mediating properties of the antibodiesspecifically disclosed, and methods of using same to, for example,reduce HIV-1 infection risk. Combinations of the antibodies, orfragments thereof, disclosed herein can also be used in the methods ofthe invention.

In certain embodiments the invention provides a bispecific antibody. Abispecific or bifunctional antibody is an artificial hybrid antibodyhaving two different heavy/light chain pairs and two different bindingsites (see, e.g., Romain Rouet & Daniel Christ “Bispecific antibodieswith native chain structure” Nature Biotechnology 32, 136-137 (2014);Byrne et al. “A tale of two specificities: bispecific antibodies fortherapeutic and diagnostic applications” Trends in Biotechnology, Volume31, Issue 11, November 2013, Pages 621-632 Songsivilai and Lachmann,Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol.148:1547-53 (1992) (and references therein)). In certain embodiments thebispecific antibody is a whole antibody of any isotype. In otherembodiments a bispecific fragment, for example but not limited to F(ab)₂fragment. In some embodiments, the bispecific antibodies do not includeFc portion, which makes these diabodies relatively small in size andeasy to penetrate tissues.

In certain embodiments, the bispecific antibodies could include Fcregion. Fc bearing diabodies, for example but not limited to Fc bearingDARTs are heavier, and could bind neonatal Fc receptor, increasing theircirculating half-life. See Garber “Bispecific antibodies rise again”Nature Reviews Drug Discovery 13, 799-801 (2014), FIG. 1a ; See US Pub20130295121, US Pub 20140099318 incorporated by reference in theirentirety. In certain embodiments, the invention encompasses diabodymolecules comprising an Fc domain or portion thereof (e.g. a CH2 domain,or CH3 domain). The Fc domain or portion thereof may be derived from anyimmunoglobulin isotype or allotype including, but not limited to, IgA,IgD, IgG, IgE and IgM. In some embodiments, the Fc domain (or portionthereof) is derived from IgG. In some embodiments, the IgG isotype isIgG1, IgG2, IgG3 or IgG4 or an allotype thereof. In some embodiments,the diabody molecule comprises an Fc domain, which Fc domain comprises aCH2 domain and CH3 domain independently selected from any immunoglobulinisotype (i.e. an Fc domain comprising the CH2 domain derived from IgGand the CH3 domain derived from IgE, or the CH2 domain derived from IgG1and the CH3 domain derived from IgG2, etc.). In some embodiments, the Fcdomain may be engineered into a polypeptide chain comprising the diabodymolecule of the invention in any position relative to other domains orportions of the polypeptide chain (e.g., the Fc domain, or portionthereof, may be c-terminal to both the VL and VH domains of thepolypeptide of the chain; may be n-terminal to both the VL and VHdomains; or may be N-terminal to one domain and c-terminal to another(i.e., between two domains of the polypeptide chain)).

Other modification s of the bispecific molecules are contemplated toincrease the half-life of the bispecific molecules. In some embodiments,these modifications include addition of a polypeptide portion of a serumbinding protein. See US20100174053 A1, incorporated by reference.

In some embodiments, the Fc variants of the bispecific molecules of theinvention are expected to have increased serum half-life compared to thenon-Fc variants. Skilled artisan can readily carry out various assays,including pharmacokinetic studies, to determine the half-life of thesemolecules.

In some embodiments, the invention encompasses polypeptide chains, eachof which polypeptide chains comprise a VH and VL domain, comprising CDRsas described herein. In certain embodiments, the VL and VH domainscomprising each polypeptide chain have the same specificity, and themultimer molecule is bivalent and monospecific. In other embodiments,the VL and VH domains comprising each polypeptide chain have differingspecificity and the multimer is bivalent and bispecific.

In some embodiments, the polypeptide chains in multimers furthercomprise an Fc domain. Dimerization of the Fc domains leads to formationof a diabody molecule that exhibits immunoglobulin-like functionality,i.e., Fc mediated function (e.g., Fc-Fc.gamma.R interaction, complementbinding, etc.).

Formation of bispecific molecule as described supra requires theinteraction of differing polypeptide chains. Such interactions aredifficult to achieve with efficiency within a single cell recombinantproduction system, due to the many variants of potential chainmispairings. One solution to increase the probability of mispairings, isto engineer “knobs-into-holes” type mutations into the desiredpolypeptide chain pairs. Such mutations favor heterodimerization overhomodimerization. For example, with respect to Fc-Fc-interactions, anamino acid substitution (preferably a substitution with an amino acidcomprising a bulky side group forming a ‘knob’, e.g., tryptophan) can beintroduced into the CH2 or CH3 domain such that steric interference willprevent interaction with a similarly mutated domain and will obligatethe mutated domain to pair with a domain into which a complementary, oraccommodating mutation has been engineered, i.e., ‘the hole’ (e.g., asubstitution with glycine). Such sets of mutations can be engineeredinto any pair of polypeptides comprising the diabody molecule, andfurther, engineered into any portion of the polypeptides chains of thepair. Methods of protein engineering to favor heterodimerization overhomodimerization are well known in the art, in particular with respectto the engineering of immunoglobulin-like molecules, and are encompassedherein (see e.g., Ridgway et al. (1996) “‘Knobs-Into-Holes’ EngineeringOf Antibody CH3 Domains For Heavy Chain Heterodimerization,” ProteinEngr. 9:617-621, Atwell et al. (1997) “Stable Heterodimers FromRemodeling The Domain Interface Of A Homodimer Using A Phage DisplayLibrary,” J. Mol. Biol. 270: 26-35, and Xie et al. (2005) “A New FormatOf Bispecific Antibody: Highly Efficient Heterodimerization, ExpressionAnd Tumor Cell Lysis,” J. Immunol. Methods 296:95-101; each of which ishereby incorporated herein by reference in its entirety).

The invention also encompasses diabody molecules comprising variant Fcor portion thereof), which variant Fc domain comprises at least oneamino acid modification (e.g. substitution, insertion deletion) relativeto a comparable wild-type Fc domain or hinge-Fc domain (or portionthereof). Molecules comprising variant Fc domains or hinge-Fc domains(or portion thereof) (e.g., antibodies) normally have altered phenotypesrelative to molecules comprising wild-type Fc domains or hinge-Fcdomains or portions thereof. The variant phenotype may be expressed asaltered serum half-life, altered stability, altered susceptibility tocellular enzymes or altered effector function as assayed in an NKdependent or macrophage dependent assay. Fc domain variants identifiedas altering effector function are known in the art. For exampleInternational Application WO04/063351, U.S. Patent ApplicationPublications 2005/0037000 and 2005/0064514.

The bispecific diabodies of the invention can simultaneously bind twoseparate and distinct epitopes. In certain embodiments the epitopes arefrom the same antigen. In other embodiments, the epitopes are fromdifferent antigens. In non-limiting embodiments a at least one epitopebinding site is specific for a determinant expressed on an immuneeffector cell (e.g. CD3, CD16, CD32, CD64, etc.) which are expressed onT lymphocytes, natural killer (NK) cells or other mononuclear cells. Inone embodiment, the diabody molecule binds to the effector celldeterminant and also activates the effector cell. In this regard,diabody molecules of the invention may exhibit Ig-like functionalityindependent of whether they further comprise an Fc domain (e.g., asassayed in any effector function assay known in the art or exemplifiedherein).

In certain embodiments, the bispecific antibody comprises an HIVenvelope binding fragment, for example but not limited to an HIVenvelope binding fragment from any of the antibodies described herein.In other embodiments, the bispecific antibody further comprises a secondantigen-interaction-site/fragment. In other embodiments, the bispecificantibody further comprises at least one effector domain.

In certain embodiments the bispecific antibodies engage cells forAntibody-Dependent Cell-mediated Cytotoxicity (ADCC). In certainembodiments the bispecific antibodies engage natural killer cells,neutrophil polymorphonuclear leukocytes, monocytes and macrophages. Incertain embodiments the bispecific antibodies are T-cell engagers. Incertain embodiments, the bispecific antibody comprises an HIV envelopebinding fragment and CD3 binding fragment. Various CD3 antibodies areknown in the art. See for example U.S. Pat. No. 8,784,821, and UnitedStates Patent Publications No. 2014-0099318 providing various disclosureon various CD3 antibodies, which disclosure in incorporated by referencein its entirety. In certain embodiments, the bispecific antibodycomprises an HIV envelope binding fragment and CD16 binding fragment.

In certain embodiments the invention provides antibodies with dualtargeting specificity. In certain aspects the invention providesbi-specific molecules that are capable of localizing an immune effectorcell to an HIV-1 envelope expressing cell, so as facilitate the killingof the HIV-1 envelope expressing cell. In this regard, bispecificantibodies bind with one “arm” to a surface antigen on target cells, andwith the second “arm” to an activating, invariant component of the Tcell receptor (TCR) complex. The simultaneous binding of such anantibody to both of its targets will force a temporary interactionbetween target cell and T cell, causing activation of any cytotoxic Tcell and subsequent lysis of the target cell. Hence, the immune responseis re-directed to the target cells and is independent of peptide antigenpresentation by the target cell or the specificity of the T cell aswould be relevant for normal MHC-restricted activation of CTLs. In thiscontext it is crucial that CTLs are only activated when a target cell ispresenting the bispecific antibody to them, i.e. the immunologicalsynapse is mimicked. Particularly desirable are bispecific antibodiesthat do not require lymphocyte preconditioning or co-stimulation inorder to elicit efficient lysis of target cells.

In certain embodiments, the invention provides antibodies or fragmentscomprising a CDR(s) of the VH and/or VL chains, or VH and/or VL chainsof the inventive antibodies, as the HIV-1 binding arm(s) of a bispecificmolecules, e.g. but not limited to DARTS, or toxin labeled HIV-1 bindingmolecules.

In certain embodiments, such bispecific molecules comprise one portionwhich targets HIV-1 envelope and a second portion which binds a secondtarget. In certain embodiments, the first portion comprises VH and VLsequences, or CDRs from the antibodies described herein. In certainembodiments, the second target could be, for example but not limited toan effector cell. In certain embodiments the second portion is a T-cellengager. In certain embodiments, the second portion comprises asequence/paratope which targets CD3. In certain embodiments, the secondportion is an antigen-binding region derived from a CD3 antibody,optionally a known CD3 antibody. In certain embodiments, the anti-CDantibody induce T cell-mediated killing. In certain embodiments, thebispecific antibodies are whole antibodies. In other embodiments, thedual targeting antibodies consist essentially of Fab fragments. In otherembodiments, the dual targeting antibodies comprise a heavy chainconstant region (CH1). In certain embodiments, the bispecific antibodydoes not comprise Fc region. In certain embodiments, the bispecificantibodies have improved effector function. In certain embodiments, thebispecific antibodies have improved cell killing activity. Variousmethods and platforms for design of bispecific antibodies are known inthe art. See for example US Pub. 20140206846, US Pub. 20140170149,20100174053, US Pub. 20090060910, US Pub 20130295121, US Pub.20140099318, US Pub. 20140088295 which contents are herein incorporatedby reference in their entirety.

In certain embodiments the invention provides human, humanized and/orchimeric antibodies. Methods to construct such antibodies are well knownin the art.

In certain aspects the invention provides use of the antibodies of theinvention, including bispecific antibodies, in methods of treating andpreventing HIV-1 infection in an individual, comprising administering tothe individual a therapeutically effective amount of a compositioncomprising the antibodies of the invention in a pharmaceuticallyacceptable form. In certain embodiment, the methods include acomposition which includes more than one HIV-1 targeting antibody. Incertain embodiments, the HIV-1 targeting antibodies in such combinationbind different epitopes on the HIV-1 envelope. In certain embodiments,such combinations of bispecific antibodies targeting more than one HIV-1epitope provide increased killing of HIV-1 infected cells. In otherembodiments, such combinations of bispecific antibodies targeting morethan one HIV-1 epitope provide increased breadth in recognition ofdifferent HIV-1 subtypes.

The invention also includes variants of the antibodies (and fragments)disclosed herein, including variants that retain the ability to bind torecombinant Env protein, the ability to bind to the surface ofvirus-infected cells and/or ADCC-mediating properties of the antibodiesspecifically disclosed, and methods of using same to, for example,reduce HIV-1 infection risk. Combinations of the antibodies, orfragments thereof, disclosed herein can also be used in the methods ofthe invention.

Homologs and variants of a VL or a VH of an antibody that specificallybinds a polypeptide are typically characterized by possession of atleast about 75%, for example at least about 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over thefull length alignment with the amino acid sequence of interest. Proteinswith even greater similarity to the reference sequences will showincreasing percentage identities when assessed by this method, such asat least 80%, at least 85%, at least 90%, at least 95%, at least 98%, orat least 99% sequence identity. When less than the entire sequence isbeing compared for sequence identity, homologs and variants willtypically possess at least 80% sequence identity over short windows of10-20 amino acids, and may possess sequence identities of at least 85%or at least 90% or 95% depending on their similarity to the referencesequence. Methods for determining sequence identity over such shortwindows are available at the NCBI website on the internet. One of skillin the art will appreciate that these sequence identity ranges areprovided for guidance only; it is entirely possible that stronglysignificant homologs could be obtained that fall outside of the rangesprovided.

In certain embodiments, the invention provides antibodies which are 99%,98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%,84%, 83%, 82%, 81%, 80% identical to the VH and VL amino acid sequencesof the antibodies described herein and still maintain their epitopebinding breadth and/or potency. In certain embodiments, the inventionprovides antibodies which are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%,91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% identical tothe CDR1, 2, and/or 3 of VH and CDR1, 2, and/or 3 VL amino acidsequences of the antibodies described herein and still maintain theirepitope binding breadth and/or potency.

In another aspect, the invention provides Fc bearing bispecificmolecules. In some embodiments, the third Domain of one or both of thepolypeptides may additionally comprises the sequence of a CH2-CH3Domain, such that complexing of the diabody polypeptides forms an FcDomain that is capable of binding to the Fc receptor of cells (such as Blymphocytes, dendritic cells, natural killer cells, macrophages,neutrophils, eosinophils, basophils and mast cells) (FIGS. 8B-8C). Manyvariations of such molecules have been described (see, e.g., UnitedStates Patent Publications No. 2014-0099318; 2013-0295121; 2010-0174053and 2009-0060910; European Patent Publication No. EP 2714079; EP2601216; EP 2376109; EP 2158221 and PCT Publications No. WO 2015/026894;WO 2015/026892; WO 2015/021089; WO 2014/159940; WO 2012/162068; WO2012/018687; WO 2010/080538), the content of each of these publicationsin herein incorporated by reference in its entirety.

In some embodiments, these Fc-bearing DARTs may comprise threepolypeptide chains. The first polypeptide of such a diabody containsthree Domains: (i) a VL1-containing Domain, (ii) a VH2-containingDomain, (iii) a Domain that promotes heterodimerization and covalentbonding with the diabody's first polypeptide chain and (iv) a Domaincontaining a CH2-CH3 sequence. The second polypeptide of such DART™contains: (i) a VL2-containing Domain, (ii) a VH1-containing Domain and(iii) a Domain that promotes heterodimerization and covalent bondingwith the diabody's first polypeptide chain. The third polypeptide ofsuch DART™ comprises a CH2-CH3 sequence. Thus, the first and secondpolypeptide chains of such DART™ associate together to form a VL1/VH1binding site that is capable of binding to the epitope, as well as aVL2/VH2 binding site that is capable of binding to the second epitope.The first and second polypeptides are bonded to one another through adisulfide bond involving cysteine residues in their respective thirdDomains. Notably, the first and third polypeptide chains complex withone another to form an Fc Domain that is stabilized via a disulfidebond. Such diabodies have enhanced potency. Such Fc-bearing DARTs™ mayhave either of two orientations (Table 1):

TABLE 1 First 3^(rd) Chain NH₂—CH2—CH3—COOH Orientation 1^(st) ChainNH₂-VL1-VH2-Heterodimer Promoting Domain-CH2—CH3—COOH 2^(nd) ChainNH₂-VL2-VH1-Heterodimer Promoting Domain-COOH 3^(rd) ChainNH₂—CH2—CH3—COOH Second 1^(st) Chain NH₂—CH2—CH3- VL1-VH2-HeterodimerOrientation Promoting Domain-COOH 2^(nd) Chain NH₂-VL2-VH1-HeterodimerPromoting Domain-COOH

HIV×CD3 bi-specific monovalent Fc diabodies that are composed of threepolypeptide chains which associate with one another to form one bindingsite specific for an epitope of HIV and one binding site specific for anepitope of CD3 (see, FIG. 8B-8C), so as to be capable of simultaneouslybinding to HIV and to CD3. Thus, such diabodies bind to a “firstantigen,” which may be either CD3 or HIV, and a “second antigen,” whichis HIV when the first epitope is CD3, and is CD3 when the first epitopeis HIV.

As shown in FIG. 8B, the first of such three polypeptide chains willcontain, in the N-terminal to C-terminal direction, an N-terminus, theAntigen-Binding Domain of a Light Chain Variable Domain (VL) of a“first” antigen (either CD3 or HIV), the Antigen-Binding Domain of aHeavy Chain Variable Domain (VH) of a second antigen (HIV, if the firstantigen was CD3; CD3, if the first antigen was HIV), aHeterodimerization-Promoting Domain, and a C-terminus. An interveninglinker peptide (Linker 1) separates the Antigen-Binding Domain of theLight Chain Variable Domain from the Antigen-Binding Domain of the HeavyChain Variable Domain. In non-limiting embodiments, the Antigen-BindingDomain of the Heavy Chain Variable Domain is linked to theHeterodimerization-Promoting Domain by an intervening linker peptide(Linker 2). In the case of an HIV×CD3 bi-specific monovalent Fc diabody,the C-terminus of the Heterodimerization-Promoting Domain is linked tothe CH2-CH3 domains of an Fc region (“Fc Domain”) by an interveninglinker peptide (Linker 3) or by an intervening spacer-linker peptide(Spacer-Linker 3). In non-limiting embodiments, the first of the threepolypeptide chains will thus contain, in the N-terminal to C-terminaldirection: VL_(First Antigen)-Linker 1-VH_(Second Antigen)-Linker 2Heterodimerization-Promoting Domain-Spacer-Linker 3-Fc Domain.

Alternatively, as shown in FIG. 8C, the first of such three polypeptidechains will contain, in the N-terminal to C-terminal direction, anN-terminus, Linker 3, the CH2-CH3 domains of an Fc region (“Fc Domain”),an intervening spacer peptide (Linker 4), having, for example the aminoacid sequence: APSSS (SEQ ID NO:39) or the amino acid sequence APSSSPME(SEQ ID NO:40), the Antigen-Binding Domain of a Light Chain VariableDomain (VL) of the first antigen (either CD3 or HIV), theAntigen-Binding Domain of a Heavy Chain Variable Domain (VII) of thesecond antigen (HIV, if the first antigen was CD3; CD3, if the firstantigen was HIV), a Heterodimerization-Promoting Domain, and aC-terminus. An intervening linker peptide (Linker 1) separates theAntigen-Binding Domain of the Light Chain Variable Domain from theAntigen-Binding Domain of the Heavy Chain Variable Domain. Innon-limiting embodiments, the Antigen-Binding Domain of the Heavy ChainVariable Domain is linked to the Heterodimerization-Promoting Domain byan intervening linker peptide (Linker 2). In non-limiting embodiments,the first of the three polypeptide chains will thus contain, in theN-terminal to C-terminal direction: Linker 3-Fc Domain-Linker4-VL_(First Antigen)-Linker 1-VH_(Second Antigen)-Linker2-Heterodimerization-Promoting Domain.

In non-limiting embodiments, the second of such three polypeptide chainswill contain, in the N-terminal to C-terminal direction, an N-terminus,the Antigen-Binding Domain of a Light Chain Variable Domain (VL) of thesecond antigen, the Antigen-Binding Domain of a Heavy Chain VariableDomain (VH) of the first antigen, a Heterodimerization-Promoting Domainand a C-terminus. An intervening linker peptide (Linker 1) separates theAntigen-Binding Domain of the Light Chain Variable Domain from theAntigen-Binding Domain of the Heavy Chain Variable Domain. Innon-limiting embodiments, the Antigen-Binding Domain of the Heavy ChainVariable Domain is linked to the Heterodimerization-Promoting Domain byan intervening linker peptide (Linker 2). In non-limiting embodiments,the second of the three polypeptide chains will thus contain, in theN-terminal to C-terminal direction: VL_(Second Antigen)-Linker1-VH_(First Antigen)-Linker 2-Heterodimerization-Promoting Domain.

In non-limiting embodiments, the third of such three polypeptide chainswill contain the linker peptide (Linker 3) and the CH2-CH3 domains of anFc region (“Fc Domain”).

The bispecific molecules of the invention contemplate designs withvarious linkers to separate the different domain comprised in thepolypeptide chains. Specific non-limiting embodiments of the linkers aredisclosed herein. Other linkers can be readily determined. Someadditional examples of linkers are disclosed in US Pub 20100174053,incorporated by reference in its entirety.

The Antigen-Binding Domain of the Light Chain Variable Domain of thefirst polypeptide chain interacts with the Antigen-Binding Domain of theHeavy Chain Variable Domain of the second polypeptide chain in order toform a functional antigen-binding site that is specific for the firstantigen (i.e., either HIV or CD3). Likewise, the Antigen-Binding Domainof the Light Chain Variable Domain of the second polypeptide chaininteracts with the Antigen-Binding Domain of the Heavy Chain VariableDomain of the first polypeptide chain in order to form a secondfunctional antigen-binding site that is specific for the second antigen(i.e., either CD3 or HIV, depending upon the identity of the firstantigen). Thus, the selection of the Antigen-Binding Domain of the LightChain Variable Domain and the Antigen-Binding Domain of the Heavy ChainVariable Domain of the first and second polypeptide chains arecoordinated, such that the two polypeptide chains collectively compriseAntigen-Binding Domains of light and Heavy Chain Variable Domainscapable of binding to HIV and CD3.

The Fc Domain of the HIV×CD3 bi-specific monovalent Fc diabodies of thepresent invention may be either a complete Fc region (e.g., a completeIgG Fc region) or only a fragment of a complete Fc region. Although theFc Domain of the bi-specific monovalent Fc diabodies of the presentinvention may possess the ability to bind to one or more Fc receptors(e.g., FcγR(s)), In non-limiting embodiments such Fc Domain will causereduced binding to FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B),FcγRIIIA (CD16a) or FcγRIIIB (CD16b) (relative to the binding exhibitedby a wild-type Fc region) or will substantially eliminate the ability ofsuch Fc Domain to bind to such receptor(s). The Fc Domain of thebi-specific monovalent Fc diabodies of the present invention may includesome or all of the CH2 Domain and/or some or all of the CH3 Domain of acomplete Fc region, or may comprise a variant CH2 and/or a variant CH3sequence (that may include, for example, one or more insertions and/orone or more deletions with respect to the CH2 or CH3 domains of acomplete Fc region). The Fc Domain of the bi-specific monovalent Fcdiabodies of the present invention may comprise non-Fc polypeptideportions, or may comprise portions of non-naturally complete Fc regions,or may comprise non-naturally occurring orientations of CH2 and/or CH3domains (such as, for example, two CH2 domains or two CH3 domains, or inthe N-terminal to C-terminal direction, a CH3 Domain linked to a CH2Domain, etc.).

In non-limiting embodiments the first and third polypeptide chains ofthe HIV×CD3 bi-specific monovalent Fc diabodies of the present inventioneach comprise CH2-CH3 domains that complex together to form animmunoglobulin (IgG) Fc Domain. The amino acid sequence of the CH2-CH3domain of human IgG1 is (SEQ ID NO:41):

APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLHQDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYTLPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK

Thus the CH2 and/or CH3 Domains of the first and third polypeptidechains may both be composed of SEQ ID NO:41, or a variant thereof.

In non-limiting embodiments the CH2-CH3 domains of the first and thirdpolypeptide chains of the HIV×CD3 bi-specific monovalent Fc diabodies ofthe present invention to exhibit decreased (or substantially no) bindingto FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a) orFcγRIIIB (CD16b) (relative to the binding exhibited by the wild-type Fcregion (SEQ ID NO:41). Fc variants and mutant forms capable of mediatingsuch altered binding are well known in the art and include amino acidsubstitutions at positions 234 and 235, a substitution at position 265or a substitution at position 297 (see, for example, U.S. Pat. No.5,624,821, herein incorporated by reference). In non-limitingembodiments the CH2-CH3 Domain of the first and/or third polypeptidechains of the HIVx CD3 bi-specific monovalent Fc diabodies of thepresent invention include a substitution at position 234 with alanineand 235 with alanine.

The CH2 and/or CH3 Domains of the first and third polypeptide chainsneed not be identical in sequence, and advantageously are modified tofoster complexing between the two polypeptide chains. For example, anamino acid substitution (for example a substitution with an amino acidcomprising a bulky side group forming a ‘knob’, e.g., tryptophan) can beintroduced into the CH2 or CH3 Domain such that steric interference willprevent interaction with a similarly mutated domain and will obligatethe mutated domain to pair with a domain into which a complementary, oraccommodating mutation has been engineered, i.e., ‘the hole’ (e.g., asubstitution with glycine). Such sets of mutations can be engineeredinto any pair of polypeptides comprising the bi-specific monovalent Fcdiabody molecule, and further, engineered into any portion of thepolypeptides chains of the pair. Methods of protein engineering to favorheterodimerization over homodimerization are well known in the art, inparticular with respect to the engineering of immunoglobulin-likemolecules, and are encompassed herein (see e.g., Ridgway et al. (1996)“Knobs-Into-Holes' Engineering Of Antibody CH3 Domains For Heavy ChainHeterodimerization,” Protein Engr. 9:617-621, Atwell et al. (1997)“Stable Heterodimers From Remodeling The Domain Interface Of A HomodimerUsing A Phage Display Library,” J. Mol. Biol. 270: 26-35, and Xie et al.(2005) “A New Format Of Bispecific Antibody: Highly EfficientHeterodimerization, Expression And Tumor Cell Lysis,” J. Immunol.Methods 296:95-101; each of which is hereby incorporated herein byreference in its entirety). In non-limiting embodiments the ‘knob’ isengineered into the CH2-CH3 Domains of the first polypeptide chain andthe ‘hole’ is engineered into the CH2-CH3 Domains of the thirdpolypeptide chain. Thus, the ‘knob’ will help in preventing the firstpolypeptide chain from homodimerizing via its CH2 and/or CH3 Domains. Innon-limiting embodiments, as the third polypeptide chain contains the‘hole’ substitution it will heterodimerize with the first polypeptidechain as well as homodimerize with itself. In non-limiting embodiments aknob is created by modifying a native IgG Fc Domain to contain themodification T366W. In non-limiting embodiments a hole is created bymodifying a native IgG Fc Domain to contain the modification T366S,L368A and Y407V. To aid in purifying the third polypeptide chainhomodimer from the final bi-specific monovalent Fc diabody comprisingthe first, second and third polypeptide chains, the protein A bindingsite of the CH2 and CH3 Domains of the third polypeptide chain ismutated by amino acid substitution at position 435 (H435R). Thus, thethird polypeptide chain homodimer will not bind to protein A, whereasthe bi-specific monovalent Fc diabody will retain its ability to bindprotein A via the protein A binding site on the first polypeptide chain.

In non-limiting embodiments a sequence for the CH2 and CH3 Domains ofthe first polypeptide chain of the HIV×effector (e.g.CD3) bi-specificmonovalent Fc diabodies of the present invention will have the“knob-bearing” sequence (SEQ ID NO:42):

APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLHQDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT  LPPSREEMTK NQVSL W C LVK GFYPSDIAVE WESNGQPENN  YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHN H YTQKS LSLSPGK

In non-limiting embodiments a sequence for the CH2 and CH3 Domains ofthe third polypeptide chain of the HIV×effector (e.g. CD3) bi-specificmonovalent Fc diabodies of the present invention will have the“hole-bearing” sequence (SEQ ID NO:43):

APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLHQDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT  LPPSREEMTK NQVSL S C AVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFL V SKL TVDKSRWQQG NVFSCSVMHEALHN R YTQKS LSLSPGK

As will be noted, the CH2-CH3 Domains of SEQ ID NO:42 and SEQ ID NO:43include a substitution at position 234 with alanine and 235 withalanine, and thus form an Fc Domain exhibit decreased (or substantiallyno) binding to FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA(CD16a) or FcγRIIIB (CD16b) (relative to the binding exhibited by thewild-type Fc region (SEQ ID NO:41).

In non-limiting embodiments a the first polypeptide chain will have a“knob-bearing” CH2-CH3 sequence, such as that of SEQ ID NO:42. However,as will be recognized, a “hole-bearing” CH2-CH3 Domain (e.g., SEQ IDNO:43) could be employed in the first polypeptide chain, in which case,a “knob-bearing” CH2-CH3 Domain (e.g., SEQ ID NO:42) would be employedin the third polypeptide chain.

In non-limiting embodiments, the Fc domain can be modified by amino acidsubstitution to increase binding to the neonatal Fc receptor andtherefore the half-life of the antibody when administered to a subject.The Fc domain can be an IgA, IgM, IgD, IgE or IgG Fc domain. The Fcdomain can be an optimized Fc domain, as described in U.S. PublishedPatent Application No. 20100093979, incorporated herein by reference. Incertain embodiments the antibodies comprise amino acid alterations, orcombinations thereof, for example in the Fc region outside of epitopebinding, which alterations can improve their properties. Various Fcmodifications are known in the art. Amino acid numbering is according tothe EU Index in Kabat. In some embodiments, the invention contemplatesantibodies comprising mutations that affect neonatal Fc receptor (FcRn)binding, antibody half-life, and localization and persistence ofantibodies at mucosal sites. See e.g. Ko S Y et al., Nature 514: 642-45,2014, at FIG. 1a and citations therein; Kuo, T. and and Averson, V.,mAbs 3(5): 422-430, 2011, at Table 1, US Pub 20110081347 (an asparticacid at Kabat residue 288 and/or a lysine at Kabat residue 435), US Pub20150152183 for various Fc region mutation, incorporated by reference intheir entirety.

In certain embodiments, the antibodies comprise AAAA substitution in andaround the Fc region of the antibody that has been reported to enhanceADCC via NK cells (AAA mutations) containing the Fc region aa of S298Aas well as E333A and K334A (Shields R I et al JBC, 276: 6591-6604, 2001)and the 4^(th) A (N434A) is to enhance FcR neonatal mediated transportof the IgG to mucosal sites (Shields R I et al. ibid). Other antibodymutations have been reported to improve antibody half-life or functionor both and can be incorporated in sequences of the antibodies. Theseinclude the DLE set of mutations (Romain G, et al. Blood 124: 3241,2014), the LS mutations M428L/N434S, alone or in a combination withother Fc region mutations, (Ko S Y et al. Nature 514: 642-45, 2014, atFIG. 1a and citations therein; Zlevsky et al., Nature Biotechnology,28(2): 157-159, 2010; US Pub 20150152183); the YTE Fc mutations (RobbieG et al Antimicrobial Agents and Chemotherapy 12: 6147-53, 2013) as wellas other engineered mutations to the antibody such as QL mutations, IHHmutations (Ko S Y et al. Nature 514: 642-45, 2014, at FIG. 1a andrelevant citations; See also Rudicell R et al. J. Virol 88: 12669-82,201). In some embodiments, modifications, such as but not limited toantibody fucosylation, may affect interaction with Fc receptors (Seee.g. Moldt, et al. JVI 86(11): 66189-6196, 2012). In some embodiments,the antibodies can comprise modifications, for example but not limitedto glycosylation, which reduce or eliminate polyreactivity of anantibody. See e.g. Chuang, et al. Protein Science 24: 1019-1030, 2015.In some embodiments the antibodies can comprise modifications in the Fcdomain such that the Fc domain exhibits, as compared to an unmodified Fcdomain enhanced antibody dependent cell mediated cytotoxicity (ADCC);increased binding to Fc.gamma.RIIA or to Fc.gamma.RIIIA; decreasedbinding to Fc.gamma.RIIB; or increased binding to Fc.gamma.RIIB. Seee.g. US Pub 20140328836.

The antibodies, and fragments thereof, described above can be formulatedas a composition (e.g., a pharmaceutical composition). Suitablecompositions can comprise the ADCC-mediating antibody (or antibodyfragment) dissolved or dispersed in a pharmaceutically acceptablecarrier (e.g., an aqueous medium). The compositions can be sterile andcan be in an injectable form (e.g. but not limited to a form suitablefor intravenous injection, or intramascular injection). The antibodies(and fragments thereof) can also be formulated as a compositionappropriate for topical administration to the skin or mucosa. Suchcompositions can take the form of liquids, ointments, creams, gels andpastes. The antibodies (and fragments thereof) can also be formulated asa composition appropriate for intranasal administration. The antibodies(and fragments thereof) can be formulated so as to be administered as apost-coital douche or with a condom. Standard formulation techniques canbe used in preparing suitable compositions.

The antibody (and fragments thereof), for example the ADCC-mediatingantibodies, described herein have utility, for example, in settingsincluding but not limited to the following:

i) in the setting of anticipated known exposure to HIV-1 infection, theantibodies described herein (or fragments thereof) and be administeredprophylactically (e.g., IV, topically or intranasally) as amicrobiocide,

ii) in the setting of known or suspected exposure, such as occurs in thesetting of rape victims, or commercial sex workers, or in any homosexualor heterosexual transmission without condom protection, the antibodiesdescribed herein (or fragments thereof) can be administered aspost-exposure prophylaxis, e.g., IV or topically, and

iii) in the setting of Acute HIV-1 infection (AHI), the antibodiesdescribed herein (or fragments thereof) can be administered as atreatment for AHI to control the initial viral load, or for theelimination of virus-infected CD4 T cells.

In accordance with the invention, the ADCC-mediating antibody (orantibody fragments) described herein can be administered prior tocontact of the subject or the subject's immune system/cells with HIV-1or within about 48 hours of such contact. Administration within thistime frame can maximize inhibition of infection of vulnerable cells ofthe subject with HIV-1.

In addition, various forms of the antibodies described herein can beadministered to chronically or acutely infected HIV-1 patients and usedto kill remaining virus infected cells by virtue of these antibodiesbinding to the surface of virus infected cells and being able to delivera toxin to these reservoir cells. In certain embodiments, the antibodiesof the invention can be administered in combination with latencyactivating agents, so as to activate latent reservoir of HIV-infectedcells. The expectation is that by activating latent proviral HIV DNA inresting cells, once inactive cells will start producing new virus andthey will be recognized and eliminated by the immune system.Non-limiting examples of latency activating agents are HDAC inhibitors,e,g, vorinostat, romidepsin, panobinostat, disulfiram, JQ1, bryostatin,PMA, inonomycin, or any combination thereof. See Bullen et al. NatureMedicine 20, 425-429 (2014).

Suitable dose ranges can depend on the antibody (or fragment) and on thenature of the formulation and route of administration. Optimum doses canbe determined by one skilled in the art without undue experimentation.For example, doses of antibodies in the range of 1-50 mg/kg of unlabeledor labeled antibody (with toxins or radioactive moieties) can be used.If antibody fragments, with or without toxins are used or antibodies areused that can be targeted to specific CD4 infected T cells, then lessantibody can be used (e.g., from 5 mg/kg to 0.01 mg/kg).

Antibodies of the invention and fragments thereof can be producedrecombinantly using nucleic acids comprising nucleotide sequencesencoding VH and VL sequences selected from those shown in the figuresand examples.

In certain embodiments the invention provides intact/whole antibodies.In certain embodiments the invention provides antigen binding fragmentsthereof. Typically, fragments compete with the intact antibody fromwhich they were derived for specific binding to the target includingseparate heavy chains, light chains Fab, Fab′, F(ab′).sub.2, F(ab)c,diabodies, Dabs, nanobodies, and Fv. Fragments can be produced byrecombinant DNA techniques, or by enzymatic or chemical separation ofintact immunoglobulins.

Nucleic acid sequences encoding polypeptides for the production ofbispecific antibodies with specificities as described herein can be usedto produce plasmids for stable expression of recombinant antibodies.Methods for recombinant expression and purification are known in theart. In certain embodiments of Fc, the plasmids also comprise any of thechanges to the Fc portion described herein. In some embodiments, theseare AAAA substitution in and around the Fc region of the antibody thathas been reported to enhance ADCC via NK cells (AAA mutations)containing the Fc region aa of S298A as well as E333A and K334A (ShieldsR I et al JBC, 276: 6591-6604, 2001) and the 4^(th) A (N434A) is toenhance FcR neonatal mediated transport of the IgG to mucosal sites(Shields R I et al. ibid).

In certain embodiments, the nucleic acids are optimized for recombinantexpression in a suitable host cell. In certain embodiments, the vectoris suitable for gene delivery and expression. There are numerousexpression systems available for expression of proteins including E.coli, other bacterial hosts, yeast, and various higher eukaryotic cellssuch as the COS, CHO, HeLa and myeloma cell lines.

Any suitable cell line can be used for expression of the polypeptides ofthe invention, including but not limited to CHO cells, 293T cells. Insome aspects, the invention provides nucleic acids encoding theseantibodies, expression cassettes and vectors including these nucleicacids, and isolated cells that express the nucleic acids which encodethe antibodies of the invention are also provided. The polypeptides ofthe invention can be purified by any suitable method for purification ofpolypeptides and/or antibodies.

The contents of the various publications cited throughout thespecification are incorporated by reference in their entirety.

EXAMPLES Example 1A: Construction of an HIV-1×CD3 or HIV-1×CD16Bispecific Molecules and Control Bispecific Molecules

Table 2 contains a list of bi-specific diabodies that were designed,expressed and purified. The bi-specific diabodies are heterodimers, orheterotrimer of the recited amino acid sequences. Methods for formingbi-specific diabodies are provided in WO 2006/113665, WO 2008/157379, WO2010/080538, WO 2012/018687, WO 2012/162068, WO 2012/162067, WO2014/159940, WO 2015/021089, WO 2015/026892 and WO 2015/026894.

TABLE 2 Polypeptide Chain Amino Nucleic Acid Encoding Bi-SpecificMolecule Acid Sequences Sequences HIV-1 × CD3 Bi-Specific Diabody SEQ IDNO: 9 SEQ ID NO: 10 (A32 × CD3) SEQ ID NO: 11 SEQ ID NO: 12 (Variabledomain from A32, binds to HIV-1 gp120) HIV-1 × CD3 Bi-Specific DiabodySEQ ID NO: 13 SEQ ID NO: 14 (7B2 × CD3) SEQ ID NO: 15 SEQ ID NO: 16(Variable domain from 7B2, binds to HIV-1 gp41) HIV-1 × CD3 Bi-SpecificDiabody SEQ ID NO: 17 SEQ ID NO: 18 (CH28 × CD3) SEQ ID NO: 19 SEQ IDNO: 20 (Variable domain from CH28, binds to HIV-1 gp) HIV-1 × CD3Bi-Specific Diabody SEQ ID NO: 21 SEQ ID NO: 22 (CH44 × CD3) SEQ ID NO:23 SEQ ID NO: 24 (Variable domain from CH44, binds to HIV-1 gp) HIV-1 ×CD16 Bi-Specific Diabody SEQ ID NO: 25 SEQ ID NO: 26 (7B2 × CD16) SEQ IDNO: 27 SEQ ID NO: 28 (Variable domain from 7B2, binds to HIV-1 gp41)Fluorescein × CD3 Bi-Specific Diabody SEQ ID NO: 29 (4420 × CD3) SEQ IDNO: 30 Fluorescein × CD16 Bi-Specific SEQ ID NO: 31 Diabody SEQ ID NO:32 (4420 × CD16) HIV-1 × Fluorescein Bi-Specific SEQ ID NO: 33 DiabodySEQ ID NO: 34 (7B2 × 4420) (Variable domain from 7B2, binds to HIV-1gp41) HIV-1 × Fluorescein Bi-Specific SEQ ID NO: 35 Diabody SEQ ID NO:36 (A32 × 4420) (Variable domain from A32, binds to HIV-1 gp120)Palivizumab × CD3 Bi-Specific Diabody SEQ ID NO: 37 (Palivizumab × CD3)SEQ ID NO: 38 HIV-1 × CD16 Bi-Specific Diabody SEQ ID NO: 44 (A32 ×CD16) SEQ ID NO: 45 (Variable domain from A32, binds to HIV-1 gp120)HIV-1 × CD3 Bi-Specific Diabody with SEQ ID NO: 46 Fc Domain V1 (7B2 ×CD3) SEQ ID NO: 47 (Variable domain from 7B2, binds to SEQ ID NO: 48HIV-1 gp41)

HIV-1×CD3 bi-specific diabodies are capable of simultaneously binding toHIV-1 and CD3. HIV-1×CD16 bi-specific diabodies are capable ofsimultaneously binding to HIV-1 and CD16. The control bi-specificdiabody (4420×CD3) is capable of simultaneously binding to FITC and CD3.The control bi-specific diabody (4420×CD16) is capable of simultaneouslybinding to FITC and CD16. The control bi-specific diabody (7B2×4420) iscapable of simultaneously binding to HIV-1 and FITC. The controlbi-specific diabody (A32x 4420) is capable of simultaneously binding toHIV-1 and FITC. The control bi-specific diabody (Palivizumab×CD3) iscapable of simultaneously binding to RSV and CD3.

Table 3 shows a summary of some embodiments of bispecific molecules. Theinformation in the specification can be readily used for alternativedesigns of the listed bispecific molecules, and for design of otherbispecific molecules, for example 7B2, CH27, Ch28, CH44 using CDRs, orVH and VL chains from these antibodies.

HIV × CD3 A32 × CD3 One embodiment HIV × CD3 Fc V1 HIV × CD3 FcV2 OneSEQ ID Nos: 9 One One A32 × CD3 Fc embodiment and 11 Together embodimentA32 × CD3 Fc V1 embodiment V2 Polypeptide SEQ ID NO: 9 is PolypeptidePolypeptide Chain 1 one embodiment Chain 1 Chain 1 NH2- SEQ ID NH2- SEQID NO: 78 NH2-Linker3 SEQ ID NO: 49 VL(HIV) NO: 78(FIG. 6) - VL(HIV)Linker 1 SEQ ID NO: 1 Linker 1 SEQ ID NO: 1 CH2—CH3 SEQ ID NO: 42 (knobbearing) or SEQ ID NO: 43 (hole bearing) VH(CD3) SEQ ID NO: 51 VH(CD3)SEQ ID NO: 51 Linker 4 SEQ ID NO: 39, 40 Linker 2 SEQ ID NO: 2 Linker 2SEQ ID NO: 2 VL(HIV) SEQ ID NO: 78 Heterodimer SEQ ID Nos: 3-6Heterodimer SEQ ID Nos: 3-6 Linker 1 SEQ ID NO: 1 promoting SEQ ID Nos:7 or promoting SEQ ID Nos: 7 domain 8 domain or 8 K coil or E K coil orE coil coil Linker 3 or SEQ ID NO: 49 VH (CD3) SEQ ID NO: 51 Spacer SEQID NO: 50 Linker 3 CH2—CH3 SEQ ID NO: 42 Linker 2 SEQ ID NO: 2 (knobbearing) or SEQ ID NO: 43 (hole bearing) Heterodimer SEQ ID Nos: 3-promoting 6 domain SEQ ID Nos: 7 K coil or E or 8 coil Polypeptide SEQID NO: 11 Polypeptide Polypeptide Chain 2 is one Chain 2 Chain 2embodiment NH2- SEQ ID NO: 52 NH2- SEQ ID NO: 52 NH2- SEQ ID NO: VL(CD3)VL(CD3) VL(CD3) 52 Linker 1 SEQ ID NO: 1 Linker 1 SEQ ID NO: 1 Linker 1SEQ ID NO: 1 VH(HIV1) SEQ ID NO: 77 VH(HIV1) SEQ ID NO: 77 VH(HIV) SEQID NO: 77 (FIG. 6) Linker 2 SEQ ID NO: 2 Linker 2 SEQ ID NO: 2 Linker 2SEQ ID NO: 2 Heterodimer SEQ ID Nos: 3-6 Heterodimer SEQ ID Nos: 3-6Heterodimer SEQ ID Nos: 3- promoting SEQ ID Nos: 7 or promoting SEQ IDNos: 7 promoting 6 domain 8 domain or 8 domain SEQ ID Nos: 7 K coil or EK coil or E K coil or E or 8 coil coil coil Polypeptide NONE PolypeptidePolypeptide Chain 3 Chain 3 Chain 3 NH2- Linker SEQ ID NO: 49 NH2-Linker 3 SEQ ID NO: 3 49 CH2—CH3 SEQ ID NO: 42 CH2—CH3 SEQ ID NO: 42(knob bearing) (knob bearing) or or SEQ ID NO: 43 SEQ ID NO: 43 (holebearing) (hole bearing)

Example 1C: HIV-1 Antibodies with ADCC Activity

Monoclonal Antibodies.

Five mAbs representing those directed against the HIV-1 env gp120constant region 1 (C1; n=1), CD4 binding site (CD4bs; n=3), and the gp41Cluster 1 [Pollara J. Curr. HIV Res. 2013; 11(8):378-3870]. All the mAbsare listed in Table 1. All but mAbs were generated with a sequence forthe Fc region that included amino acid substitutions according toShields et al to optimize the binding to the Fcγ-Receptor (Fcγ-R) IIIa[Shields RL J Biol Chem 2001; 276(9): 6591-6604].

A32 mAb recognizes a conformational epitope in the C1 region of HIV-1Env gp120 (Wyatt et al, J. Virol. 69:5723-5733 (1995)), could mediatepotent ADCC activity and could block a significant proportion ofADCC-mediating Ab activity detectable in HIV-1 infected individuals(Ferrari et al, J. Virol. 85:7029-7036 (2011)).

CH28 or CH44 are HIV-1 CD4 bs neutralizing antibodies.

TABLE 4 List of mAbs tested for ADCC gp120 gp41 C1 CD4bs Cluster I A32CH27 7B2 CH28 CH44 All mAbs were produced in the 3A version to optimizethe binding to the Fcγ-Receptor IIIa, but those identified by the symbol(*).

Infectious Molecular Clones (IMC).

The HIV-1 IMCs represented 22 isolates to represent those with variousdegree of susceptibility to neutralization based on testing with theA3R5 cell line. The list of the IMCs is reported in Table 5.

TABLE 5 List of IMC by HIV-1 subtype used to generate infected targetcells. A AE Bc C Q23.17 C1080.C03 SF162 MW96.5 427299 BaL CAP45 92TH023CH058 245-F3-C10 CM235 CH040 TV-1 CM244 SUMA CH0505 816763 WITO DU151YU2 DU422 1086.c

All IMCs were generated on backbone derived from NHL4-3 isolate aspreviously described [Edmonds TG. Virology. 2010; 408(1):1-13; Adachi A.J Virol. 1986; 59(2):284-291] but for the subtype AE 92TH023 that wasgenerated utilizing the backbone from the 40021 AE HIV-1 Isolate. AllIMCs expressed the Renato luciferase reporter gene and preserved allnine viral open reading frames. The Renato luciferase reporter gene wasexpressed under the control of the HIV-1 Tat gene. Upon HIV-1 infectionof the CD4+ T cells, expression of Tat during HIV-1 replication willinduce expression of the luciferase and infected cells can be easilyquantified by measure of Relative Luminescence Units.

Antibody Dependent Cellular Cytotoxicity (ADCC) Assay.

The assay was performed according to our previously published methodsusing a luciferase based platform as read-out for the cytotoxicitymediated by the mAbs [Pollara J. J Virol. 2014; 88(14):7715-7726]. Theeffector cells populations were all derived from a single donor with thecharacterized heterozygous phenotype FN for the amino acid in position158 of the Fcγ-R IIIa. The effector to target ratio was 30:1 in eachassay. The plasma from a HIV-1 infected individuals (A300) and thePalivizumab (anti-RSV) mAb were used as positive and negative control ineach assay. All the mAbs were tested together against each IMC. Thepercentage of specific killing (% SK) was calculated as previouslyreported. The results were considered positive if the % SK was >20%.

Potency and Breadth of ADCC-Mediating mAbs.

Each mAbs listed in Table 1 was tested individually against each of the22 IMCs listed in Table 2. The results have been evaluated to identifythe maximum ADCC activity as % SK independently from the concentrationat which the activity was observed. The mAbs were grouped based on theenv gp120 and gp41 regions recognized. The average of the positiveresponses for each mAb are reported in FIG. 1. The magnitude and breadthof the mAbs is summarized in Table 3. The non-neutralizing Abs directedagainst gp120 C1 and gp41 cluster 1 provided the broadest spectrum ofADCC by recognizing 21 (95%) and 20 (91%) HIV-1 isolates, respectively.The average % of specific killing (% SK) was 37% for the C1 mAbs and 34%for the gp41 cluster I mAbs. The averages of the maximum % SK of A32 and7B2 were 45 and 42, respectively. Cumulatively, the CH44 mAbrecognized<60% of the isolates tested with a range of activity between21 and 60% SK.

TABLE 6 Magnitude and breadth of ADCC-mediating mAbs A32 7B2 CH27 CH28CH44 Average 45 42 31 36 13 Max % SK Range 23-86 21-74 21-53 23-48 21-60# IMCs 21 20  8  7 14 recognized (%) (95%) (91%) (36%) (31%) (59%)

Example 2: Cell Killing by Dual Affinity Re-Targeting (DART) MoleculesA32/CD3 and 7B2/CD3

Dual affinity Re-Targeting molecules A32/CD3 (SEQ ID NOs: 9 and 11) and7B2/CD3 (SEQ ID NOs: 13 and 15) were designed and expressed. Thesemolecules include an HIV-1 binding arm generated based on the Fab ofanti-HIV-1 monoclonal antibodies (mAbs) (mAbs that have the property ofbinding to the surface of tier 2 transmitted/founder (T/F) virusinfected CD4 T cells (i.e. A32 or 7B2) [Ferrari G, J Virol. 2011;85(14):7029-7036; Pollara J. Curr. HIV Res. 2013; 11(8):378-3870], andan effector cell binding arm that can bind the CD3 (αCD3ε arm) or CD16(αCD16 h3G8 arm) receptors. Appropriate negative controls with anirrelevant binding arm [αfluorescein (4420) or αRSV] instead of theHIV-1 or effector arm have also been developed. The results presented inthis example are from experiments with the CD3-DARTs.

Luciferase-based Cytotoxicity Assay.

We optimized a method to quantify the elimination of HIV-1-infectedcells by cytotoxic CD8 T cells recruited by the DARTs that is based onthe detection of luciferase activity as final readout as previouslyreported [Pollara J. J Virol. 2014; 88(14):7715-7726]. Cryopreservedresting PBMC from normal healthy HIV-1 seronegative donors wereactivated for 72 hours with anti-human CD3 (clone OKT3;eBioscience)/anti-human CD28(clone CD28.2; BD Pharmingen). Subsequently,a CD4+ enriched cell population was obtained using magnetic beads,spinoculated in presence of the IMC representing the HIV-1 subtype AE(CM235), B (BaL), and C (1086.c) and cultured, for 72 hours. CD4+infected target cells were then plated along with resting CD8+ effectorcells at 33:1, 11:1, 3:1, and 0:1 effector to target ratios. DARTs(4420×CD3, 7B2×CD3, or A32×CD3) were added to combined cells atconcentrations ranging from 0.001 to 1000 ng/ml and incubated for 6, 24,and 48-hour time points. Combined effector and target cells withoutDARTs, uninfected cells, and target cells alone were included on eachplate for control conditions. At the end of each incubation time,Viviren substrate was added to each well and cells were analyzed on aluminometer to measure RLU values through luciferase readout. Inpresence of the cytotoxic cells of interest the elimination of infectedtarget cells was evaluated using the appropriate already publishedformula [Pollara J. J Virol. 2014; 88(14):7715-7726]. The results arereported as % SK as described for the ADCC assay.

Anti-HIV-1 DARTs-Mediated Cytotoxic Activity.

Based on the results described above, two DARTs were generated whoseanti-HIV-1 arm was the A32 and 7B2 Fab region and the effector cellbinding arm the αCD3c arm. We studied these two DART molecules for theirability to recognize and mediate the killing of infected CD4+ T cells.Leukapheresis samples obtained from HIV-1 seronegative donor wereinfected in vitro to generate the target cells as described in thematerial and method section using our previously described ADCCLuciferase-based assay to detect the cytotoxic effects of the DARTs. Wetested the two CD3-DART molecules (7B2×CD3 and A32×CD3) for theirability to redirect the cytotoxicity of resting CD8+ T cells againstsubtype B BaL, AE CM235, and C 1086.c HIV-1 IMC infected autologous CD4+T cells. We evaluated DART-mediated cytotoxicity at 6, 24, and 48 hoursafter incubation of effector and target cells at the effector-to-targetratios of 33, 11, and 3 to 1. Although cytotoxic activity was alreadyobserved after 6 hours incubation, the peak cytotoxic activity (>70% SK)was detected at 48 hours using the 33:1 E:T ratio against each HIV-1 IMC(FIG. 2). The activity of the two HIV-1 DARTs was always greater thanthe background maximum killing observed with the 4420×CD3 control DART.We also observed a dose dependent potency of the two DARTs against eachHIV-1 IMC-infected target cell population that is also reflected at thelevel of each E:T ratio, as illustrated for the BaL IMC (FIG. 3).

The difference in the potency of the two DARTs was also analyzed as theDART concentration at which 50% of specific killing (KillingConcentrations® or KC₅₀) was detected at 48 hours with E:T of 33:1. TheA32×CD3 DART KC₅₀ was always approximately one log lower than the7B2×CD3 DART KC₅₀ (FIG. 4) against each HIV-1 IMC.

These results indicated that DARTs can effectively recruit CD8+ T cellsand direct their cytotoxic activity against HIV-1 infected cells.

Example 3: A32/CD16 and 7B2/CD16 DARTs

Dual affinity Re-Targeting molecules A32/CD16 (SEQ ID NOs: 44 and 45)and 7B2/CD16 (SEQ ID NOs: 25 and 27 see Table 2) will be analyzed asdescribed in Example and 6 using Luciferase-based cytotoxicity assay andCD4+ infected target cells along with resting effector cells. For theCD16-DART assay, the effector cells are CD16+ cells, which could bepurified by removing CD3+CD20+ cells from whole PBMCs.

The Luciferase-based killing assay, described in Example 2 will be usedto examine and compare the potency and kinetics of CD16-DART-enhancedclearance of productive infection as previously proposed for theCD3-DARTs. The procedure will be the same but the negative selection ofthe effector cells will provide an enriched population of CD16+ cells.

Example 4: CH28 and CH44 DARTs

DART molecules with a HIV-1 arm having the binding specificity of CH28or CH44, and an effector cell arm targeting CD3 or CD16 will be made andtested in the Luciferase-based killing assay essentially as described inExamples 2 and 3. CH28 or CH44 are HIV-1 CD4 bs neutralizing antibodies.See U.S. Provisional Appl. No. 61/883,220 filed Sep. 27, 2013 andcorresponding PCT application. CH28/CD3 comprises SEQ ID NOs: 19 and 19.CH44/CD3 comprises SEQ ID NOs: 21 and 23.

Example 5: Combinations of CD13- and CD16-DARTs

The Luciferase-based killing assay will be used to test whether CD13-and CD16-DARTs in a combination formulation provide enhanced benefits.For each DART combination, we will utilize cells expressing the 3different Fcγ-R IIIa (CD16) phenotypes and the panel of established IMCsto test the ability of DARTs to recruit simultaneously CD3+ and CD16+effector cells. These assessments will be conducted using leukapheresissamples collected from HIV-1 seronegative donors.

All documents and other information sources cited herein are herebyincorporated in their entirety by reference.

Example 6: Dual-Affinity-Re-Targeting (DART) Proteins DirectT-Cells-Mediated Cytolysis of Latently HIV-Infected Cells

Enhancement of HIV-specific immunity is likely required to eliminatelatent HIV infection. To this aim, a novel immunotherapeutic modalityhas been developed, Dual Affinity Re-Targeting (DART) proteins that arebispecific antibody-based molecules that can bind two distinct cellsurface molecules simultaneously. Described herein are HIV×CD3 DARTsdesigned with a monovalent HIV-1 envelope (Env) binding arm, derivedfrom broadly binding, ADCC-mediating antibodies known to bind toHIV-infected target cells, that is coupled to a monovalent CD3 bindingarm designed to engage cytolytic effector T-cells. Thus, DARTs redirectpolyclonal T-cells to specifically engage with, and kill Env-expressingcells, including CD4⁺ T cells infected with different HIV-1 subtypes,thereby obviating the requirement for HIV-specific immunity. Usinglymphocytes from patients on suppressive anti-retroviral therapy (ART),DARTs mediated CD8⁺ T-cell clearance in vitro of CD4⁺ T-cellssuperinfected with the HIV-1 strain JR-CSF or infected with autologousreservoir viruses isolated from HIV-infected patient resting CD4⁺T-cells. Importantly, DARTs also mediated CD8⁺ T-cell clearance of HIVfrom resting CD4⁺ T cell cultures following induction of latent virusexpression. Combined with HIV latency reversing agents, HIV×CD3 DARTshave the potential to be effective immunotherapeutic agents to clearlatent HIV-1 reservoirs in HIV-infected individuals.

The inability of antiretroviral therapy (ART) to eradicate HIV was firstsuggested by the demonstration of latent infection of resting CD4⁺ Tcells (1), and then by the recovery of rare, integrated,replication-competent HIV from the resting CD4⁺ memory T cells ofpatients receiving potent ART (2-4). Current ART cannot eradicate HIVinfection, because these long-lived CD4⁺ T cells remain persistentlyinfected and unrecognized by the immune system, with minimal expressionof HIV genes or proteins (1, 5, 6). The persistence of quiescent HIVinfection, primarily within central memory T cells, is a major obstacleto eradication of HIV infection (2-4, 7-9).

Viral persistence is also manifest in a substantial proportion oftreated patients by very low levels of detectable viral RNA (10, 11)that represents expression of viral particles without effective roundsof new replication and does not appear to lead to drug resistance orfailure of therapy (12, 13). However, persistent viremia demonstrates aninability of the immune response to recognize and clear HIV-1 infectedcells.

Chronically infected individuals generally have rapid viral rebound whenART is withdrawn (14-16). This observation has suggested that the immunesystem in patients cannot control viremia, unless bolstered by a furtherintervention. Therapeutic immunization, even in individuals whoinitiated ART when CD4⁺ and CD8⁺ cellular immune responses remainrelatively preserved, has thus far been unsuccessful in inducingenhanced anti-HIV immunity that can restrict viremia in the absence ofART (17). Therefore, eliminating the latent pool of HIV-infected cellsthat persist despite ART, and as well, the unknown cells that are thesource of low-level viremia found in most patients despite ART, requiresnew and innovative strategies. One initial step, the disruption oflatency and the induction of viral antigen expression in cells that arelatently infected, is under intensive investigation (18, 19). However,as early progress is made in the development of latency reversing agents(LRAs), improvements in the ability to clear persistent infection mustbe sought as well.

Latently infected cells are very rare, and even if the latent reservoiris as much as 60-times larger than the typical estimates of about 1infected cell per 10⁶ resting central memory CD4+ cells (20), currentLRAs might induce proviral transcription in only a fraction of thispopulation, and the quantity of viral antigen presented might be low(21, 22). Therefore, a novel and robust immune response may be necessaryto detect and clear both cells producing low-level viremia, and inquiescently infected cells induced to leave the latent state.

Following the reactivation of latent HIV, viral antigens are presentedon the surface of the cell and thus could be targeted by antibodies orantibody-derived molecules. Proof of concept for this approach has beenprovided by immunotoxins—bifunctional chimeric proteins consisting of atargeting domain, such as an antibody or a ligand, joined to a toxineffector domain (23). Although initial clinical trials usingimmunotoxins in HIV-infected individuals failed to have sustained impacton immunological or clinical markers (24), immunotoxin 3B3-PE38 (25) hasbeen reported to reduce levels of HIV-infected cells that persistdespite ART in the BLT humanized mouse model (26).

Several monoclonal antibodies (mAbs) have been reported as capable ofrecognizing HIV-1 infected cells and engaging Fc-gamma receptor-bearingcells to mediate antibody dependent cellular cytotoxicity (ADCC) (27),such as A32 and 7B2, non-neutralizing mAbs that bind to conservedresidues in gp120 (28) and gp41(29, 30), respectively. Based on theseproperties, two Dual Affinity Re-Targeting (DART) proteins (31, 32) weregenerated in which HIV envelope targeting arms derived from the A32 and7B2 mAbs were combined with a CD3 effector arm derived from hXR32, ahumanized anti-CD3E mAb, to generate two HIV×CD3 DARTs, A32×CD3 and7B2×CD3 (FIG. 10).

Bispecific molecules that co-engage T cells with antigen-expressingtarget cells, such as DARTs and Bi-specific T-cell Engager proteins(BiTEs), have been characterized and developed largely for use inoncology (31-34). They are dependent on the engagement of both of thebinding arms to activate and redirect the cytolytic activity ofpolyclonal T-cells, in a major histocompatibility complex (MHC)independent manner, against the antigen expressing target cells (31-34).This class of bispecific molecules is effective in vivo at dosesmany-fold lower than those typically employed for mAbs (33, 34), and hasbeen shown to be clinically potent and efficacious with acceptablesafety, as evidenced by the approval of blinatumumab, a CD19×CD3 BiTE,for the treatment of relapsed or refractory B-precursor acutelymphoblastic leukemia (ALL) (35, 36). DARTs, which have inter-chaindisulfide bonds at their C-termini and are structurally compact, makingthem well suited for forming stable cell-to-cell contacts between targetand effector cells, exhibit greater potency than BiTEs in side-by-sidecomparisons (32, 37).

Disclosed herein is the ability of HIV×CD3 DARTs to redirect CD8⁺ Tcells against CD4⁺ cells infected by HIV-1, including ones infected withauthentic latent virus isolates emerging from HIV-infected patients'cells in model systems designed to mimic potential clinical HIVeradication strategies. The ability of HIV×CD3 DARTs to recognizeconserved HIV-1 antigens on infected cells and simultaneously engagereceptors on the membrane of polyclonal effector T-cells, will overcomethe need to activate pre-existing HIV-specific cytotoxic effector cells(38), thus surmounting a significant hurdle that impedes the effectiveelimination of the reservoir of infected CD4⁺ T cells.

HIV Arm Selection for DARTs.

A32 mAb binds to a conformational, CD4-inducible epitope in gp120 C1/C2(within epitope cluster A) (28, 39-41) and 7B2 mAb binds to a linearepitope in gp41 cluster I (29, 30, 42). The two mAbs were tested fortheir ability to mediate antibody dependent cell-mediated cytotoxicity(ADCC) against a panel of 22 representative HIV-1 infectious molecularclones (IMCs) of subtypes A, AE, B and C (FIG. 18). The A32 mAbrecognized 21 (95%) of the HIV-1 isolates with an average percentspecific lysis (% SL) of 43.69% (range 12-86%; FIG. 23). The 7B2 mAbrecognized 20 (91%) of the HIV-1 isolates with an average % SL of 39.58%(range 15-74%; FIG. 23). In addition to possessing breadth andefficiency in mediating ADCC—indicating epitope accessibility at thesurface of HIV-infected cells, a necessary property for HIV×CD3DARTs—the A32 and 7B2 mAbs are attractive sources for Env bindingdomains for DARTs as the residues in Env that influence binding by thesemAbs are highly conserved among all HIV-1 subtypes (FIG. 24). Based onthese properties, two HIV×CD3 DARTs were generated in which HIVtargeting arms derived from the A32 and 7B2 mAbs were combined with aCD3 effector arm derived from hXR32, a humanized anti-CD3E mAb (FIGS.10A-10C). These HIV×CD3 DARTs are named A32×CD3 and 7B2×CD3. ControlDARTs with an irrelevant arm derived from an anti-FITC antibody (4420)or from palivizumab, an antibody to the respiratory syncytial virus(RSV) fusion protein antibody instead of the HIV arm (4420×CD3, RSV×CD3)or CD3 arm (A32×4420, 7B2×4420) were also generated. Control DARTs withan irrelevant arm derived from an anti-FITC antibody (4420) orpalivizumab, an antibody to the respiratory syncytial virus (RSV) fusionprotein antibody, instead of the HIV (4420×CD3 and RSV×CD3) or CD3 arm(A32×4420 and 7B2×4420) were also generated.

HIV DART Binding Properties.

A32×CD3 and 7B2×CD3 each exhibited binding to recombinant human CD3 andHIV-1 Env proteins, individually and simultaneously, as shown by ELISA(FIGS. 11A-11C). While the binding to CD3 protein was similar for bothDARTs, the magnitude of binding to JR-FL gp140 CF was greater for7B2×CD3 than for A32×CD3, likely due to the fact that the conformationalA32 epitope is highly CD4-dependent (41-44). Based on surface plasmonresonance (SPR), the equilibrium dissociation constants (K_(D)) for CD3arm binding were 3.6 and 6.1 nM for A32×CD3 and 7B2×CD3, respectively,and K_(D) for HIV arm binding was 47.7 nM for A32×CD3 using M.ConS gp140CFI, and 15.1 nM for 7B2×CD3 using JR-FL gp140 CF, respectively (FIG.19). Different Env proteins were utilized for these two DARTs in the SPRstudies, because A32×CD3 binding to JR-FL gp140 CF was inefficient and7B2×CD3 binding to M.ConS gp140 CFI, due to its lack of the gp41 clusterI sequence, was precluded.

HIV×CD3 DARTs bind to their cell surface antigens with specificity.DARTs with CD3 effector arms (A32×CD3, 7B2×CD3, 4420×CD3) bind to humanCD3⁺ T cells with similar efficiencies, whereas DARTs with the CD3 armreplaced by an irrelevant arm (A32×4420, 7B2×4420) or with twoirrelevant arms (4420×4420) do not bind (FIG. 11D). HIV×CD3 DARTs(A32×CD3, 7B2×CD3) bind efficiently to HEK293-D371 cells that expresssubtype AE CM244 Env (FIG. 11E), and similar binding activity isobserved with the A32×4420 and 7B2×4420 control DARTs (FIG. 25). Asexpected, the 4420×CD3 control DART does not bind to these cells (FIG.11E). A32×CD3 and 7B2×CD3 bind to Jurkat-522 F/Y cells, which expressboth CD3 and subtype B HXBc2 Env (45) and binding via the CD3 armpredominates as shown by the equivalence of 4420×CD3, A32×CD3 and7B2×CD3 binding. When the CD3 arm is replaced by the irrelevant 4420 armto ablate CD3 binding, low level binding to the cell surface Env isdetected with A32×4420, but not with 7B2×4420 (FIG. 11F).

HIV×CD3 DART Redirected T-Cell Killing of Env-Expressing Cell Lines andConcomitant T-Cell Activation.

Jurkat 522-F/Y is a human CD4⁺ cell line that expresses Env and servesas a model for HIV-infected CD4⁺ T cells and Jurkat-ΔKS is a controlcell line that is identical, except for a deletion/frameshift mutationin the Env gene that precludes its expression (45). These cell lineswere utilized to evaluate the ability of HIV×CD3 DARTs to mediateredirected T-cell killing of Env⁺ target cells. Target cell cytolysiswas determined by measuring lactate dehydrogenase (LDH) release with thestandard assay and the results were confirmed by luminescence (LUM)assay. As measured by LDH release assays, A32×CD3 and 7B2×CD3 redirectedhuman T cells derived from healthy donors to kill the Jurkat-522 F/Ycells in a concentration dependent manner at an E:T ratio of 10:1, andthese two HIV×DARTs exhibited similar potencies after 48 h of incubationwith fifty percent effective concentrations (EC₅₀) of 160-230 pg/mL(FIG. 12A). No DART-mediated redirected T-cell killing of Jurkat-522 F/Ycells occurred with control DARTs (4420×CD3, A32×4420, 7B2×4420) inwhich the HIV arm or CD3 arm was replaced by an irrelevant one (FIG.12A). The A32×CD3 and 7B2×CD3 DARTs did not mediate target cell killingwhen the effector T-cells were omitted (FIG. 12B) or when the targetcells lacked Env expression (FIG. 12C). These data demonstrate a strictrequirement for Env-expressing target cells and their coengagement withCD3-expressing effector cells for HIV×CD3 DART mediated cytolyticactivity.

As measured by LUM assays, A32×CD3 and 7B2×CD3 exhibited similarpotencies for redirected T-cell killing of Jurkat 522-F/Y GF cells withECso values of 140-170 pg/mL (FIG. 12D), which were close to thosemeasured with the LDH release assay, indicating consistency across thetwo different assay modalities. Moreover, with the sensitivity andspecificity of the LUM assay, DART-dependent elimination of the Env⁺target cells was nearly complete (>98%), while the 4420×CD3 control DARTmediated no cytotoxicity (FIG. 12D). HIV×CD3 DART redirected T cellkilling activity was time and E:T ratio dependent. Near completecytolysis with 7B2×CD3 was reached at 48 hours at E:T ratios of 10:1 and5:1, whereas high level cytolysis (>80%) at an E:T ratio of 1:1 wasdelayed until 72 hours (FIGS. 12E-12H), suggesting that time is thelimiting factor for the efficient elimination of target cells at lowerE:T cell ratios.

Concomitant with redirecting T-cell killing activity, the HIV×CD3 DARTsinduced T-cell activation (measured by upregulation of the activationmarker, CD25) in the presence of the Env⁺ target cells with CD25upregulated in CD8⁺ T-cells to a greater extent than in CD4⁺ T-cells(FIGS. 26A-26D). The overall data demonstrate that A32×CD3 and 7B2×CD3potently activate and redirect T cells, especially CD8⁺ T-cells, tospecifically kill Env-expressing target cells. Moreover, the killingdata confirm that both DARTs were capable of recognizing and binding toEnv antigens on the surface of a CD4⁺ cell line even though thedetection of Env binding by FACS analysis was negligible (FIG. 13F).

HIV×CD3 DARTs Bind to the Surface of HIV-Infected CD4⁺ T Cells andRedirect CD8⁺ T-Cells to Kill HIV-1 Infected CD4⁺ Cells UsingLymphocytes from HIV-1 Seronegative Donors.

The A32×4420 and 7B2×4420 DARTs were evaluated for their ability to bindand redirect the killing of CD4⁺ T cell infected with HIV-1 Infectiousmolecular clones representing the subtype AE CM235, subtype B BaL, andsubtype C 1086.0 HIV-1 isolates. Each IMC was engineered with aluciferase reporter gene to quantitatively measure the cytolysis ofinfected target cells. To assess binding to infected cell surface Env,A32×4420 and 7B2×4420 DARTs (which lack CD3 effector arms) were comparedto the parental A32 and 7B2 mAbs. Similar staining of the p24⁺(infected) CD4⁺ T cells by both HIV×CD3 DARTs independently from theHIV-1 IMC used for the infection (FIG. 27) was observed. Interestingly,staining with the A32×4420 DART recapitulated closely the staining withthe A32 mAbs; in contrast, the 7B2×4420 DART recognized>66% of the HIV-1infected cells (range 66-78%) compared to the >24% recognized by the 7B2mAb (range 24-38), suggesting that the DART has a better accessibilityto the cluster I gp41 epitope compared to the mAb (FIG. 27). Thesecondary conjugated Abs and the Palivizumab mAb utilized as controlsrecognized less than <5% HIV-1 infected CD4⁺ T cells.

The ability of A32×CD3 and 7B2×CD3 to redirect CD8⁺ T cells from HIV-1seronegative donors against autologous CD4⁺ T cells infected with thethree HIV-1 IMCs was subsequently investigated. The two HIV×CD3 DARTsredirected autologous CD8⁺ T effector cells to kill subtype B BaL (FIG.13A), subtype AE CM235 (FIG. 13B), and subtype C 1086.0 (FIG. 13C)IMC-infected CD4⁺ target cells in a concentration dependent manner,whereas the control DART (4420×CD3) was inactive. The greater potencyexhibited by A32×CD3 (EC₅₀≤1 ng/mL) compared to 7B2×CD3 (EC₅₀˜10 ng/mL)in these studies with IMC-infected CD4⁺ cells contrasts with the similarpotencies observed in the studies with Envy cell lines (FIGS. 12A-12C).DART mediated killing of the IMC-infected CD4⁺ T cells was dependent onthe presence of CD8⁺ effector cells, and no cytolytic activity wasobserved in their absence (FIGS. 13A-13C). In time course studies,DART-dependent cytolytic activity was evident at 6 hours with maximalactivity (>70% cytolysis) at 48 hours (FIGS. 13D-13F).

To gain insight into the frequency of effector T cells recruited by theDARTs to kill HIV-1 infected target cells, the ability of DARTs toinduce degranulation of the CD8⁺ T cells obtained from 5 HIV-1seronegative donors when co-incubated with autologous HIV-1 BaLIMC-infected CD4⁺ cells under the same conditions used to detectcytolytic activity was assessed. The example of the gating strategyadopted for data analysis is illustrated in FIGS. 14A-14G. The meanfrequency of Live/CD3⁺/CD8⁺/CD107⁺ cells (FIG. 14H) under controlconditions (absence of HIV×CD3 DART or presence of control DART) was0.38% (standard deviation 0.10%; range 0.24-0.51), which increased to anaverage 3.53% (range 1.5-6.9%) or 18.23% (range 12.30-23.35%) in thepresence of 1 ng/mL 7B2×CD3 or A32×CD3, respectively. The datademonstrate that HIV×CD3 DARTs can specifically induce degranulation ofresting CD8⁺ T cells in the presence of Env-expressing target cells(autologous HIV-1-infected CD4⁺ T cells).

HIV×CD3 DART Redirected CD8⁺ T Cell Killing Activity Against JR-CSFInfected Cells from Seronegative Donors.

A viral clearance assay measuring HIV gag p24 antigen production wasutilized as an alternative method to assess DART redirected T cellkilling activity. CD4⁺ cells from healthy donors were superinfected withthe HIV-1 Glade B clone JR-CSF and incubated with autologous CD8⁺ Tcells at an E:T ratio of 1:1 in the absence or presence of 100 ng/mLDARTs for 7 days. In experiments with two different donors, addition ofthe control DART (4420×CD3) did not significantly reduce p24 productioncompared to incubations performed in the absence of DARTs, whereasaddition of A32×CD3 or 7B2×CD3 significantly reduced p24 production to asimilar extent (by 72-96% or 87-99% respectively; p<0.01 Student T test;FIGS. 15A-15B). The viral clearance assay was also conducted in thepresence of integrase and non-nucleoside reverse transcriptaseinhibitors once infection was established, at the time of addition ofeffector cells and DARTs, to block further rounds of infection. Whenantiretrovirals (ARVs) were included in the assay, A32×CD3 and 7B2×CD3still mediated a trend towards reduction in p24 production, althoughthis did not reach statistical significance likely due to low levels ofbaseline p24 production with the antiretrovirals (FIG. 15C), suggestingthat the DARTs are not acting by inhibition of virus spread but ratherthrough clearance of infected cells.

HIV×CD3 DARTs Redirect CD8⁺ T-Cells to Clear JR-CSF-Superinfected CD4⁺Cells Using Lymphocytes from Patients on Suppressive ART.

Chronic ART is characterized by dysfunctional and exhausted T cellresponses (46, 47) and thus confirmation of robust DART mediated T-cellredirected clearance activity in patient samples ex vivo is critical.The activity of HIV×CD3 DARTs in viral clearance assays with lymphocytesfrom 8 HIV-infected individuals on suppressive ART was evaluated. Allparticipants were on ART for at least 6 months at the time of study withvirus load<50 copies/mL, but otherwise exhibited diverse clinicalbackgrounds (FIG. 20).

Because T cells from HIV-1 seropositive subjects could be moresusceptible to apoptosis than those from seronegative subjects (48),whether HIV×CD3 DARTs, in the absence of target cells, might impactT-cell viability, which could confound the analysis of DART activitywith patients' cells, was evaluated. Following 7 days of culture ofeither CD4⁺ or CD8⁺ T cells from HIV-infected, ART-suppressed patientsin the presence of 100 ng/mL DART, which mimics the viral clearanceassay conditions, no decreases in T cell viability based on Annexin V/7AAD staining (FIGS. 28A-28B) was observed. Moreover, no changes inactivation markers (HLA-DR, CD25) on unstimulated CD4⁺ or CD8⁺ T cellswere observed after culture with HIV×CD3 or control DARTs (FIGS.28C-28D), suggesting that engagement of the CD3 arm alone does notactivate patients' CD8⁺ or CD4⁺ T-cells ex vivo.

Using the lymphocytes from 8 HIV patients on suppressive ART, viralclearance assays were conducted in which CD4⁺ cells were superinfectedwith HIV-1 JR-CSF (target cells) and incubated with autologous CD8⁺cells (effectors) at E:T ratios of 0:1, 1:10 or 1:1 in the absence orpresence of 100 ng/mL DARTs for 7 days. HIV×CD3 DART activity occurredeven in the absence of added CD8⁺ T cells, indicating, that under theseexperimental conditions, CD4⁺ T cells may be recruited as effectorcells; compared to control, p24 production was reduced by 0.89 log with7B2×CD3 (p<0.05), by 0.32 log with A32×CD3 (p=NS), and by 0.81 with a1:1 cocktail of both DARTs (p<0.05) (FIG. 16A). Indeed, the addition offully active DARTs led to significantly increased degranulation of CD4+T cells when in the presence of infected target cells (FIGS. 16G, 16H).The addition of CD8⁺ T cells as effectors resulted in further reductionsin p24 levels; compared to the 0.13 log reduction seen with CD8⁺ T cellsalone at an E:T of 1:10, p24 production was reduced by 1.2 log with7B2×CD3 (p<0.05), by 0.6 log with A32×CD3 (p=NS), and by 1.8 log with acocktail of the two DARTs (p<0.05) (FIG. 16B). Even more markedreductions were found with the higher E:T ratio of 1:1, where CD8s aloneaccounted for a 0.7 log reduction, but p24 production was reduced by 2.8log with 7B2×CD3 (p<0.05), by 1.6 log with A32×CD3 (p=NS), and by 2.8log with a cocktail of the two DARTs (p<0.05) (FIG. 16C). Significantreductions were seen even in the absence of any detectable baseline CD8T cell antiviral activity, and in three cases no virus was able to berecovered following incubation with DARTs (patient 749 with both fullyactive DARTs, and patients 720 and 725 with 7B2×CD3). The absolute HIVgag p24 antigen values are provided in FIG. 21.

HIV×CD3 DARTs redirect CD8⁺ T-cells to clear autologous reservoir virus(AR)-superinfected CD4⁺ cells using lymphocytes from patients onsuppressive ART. The ability of the DARTs to redirect T-cells againsttarget cells expressing Env sequences arising from the latent reservoirthrough the use of viral clearance assays employing autologous reservoirvirus (AR)-infected CD4⁺ target cells from 5 patients (FIGS. 16D-16F)was evaluated. Patient AR virus isolates were generated from pooledsupernatants of limiting dilution cultures of mitogen stimulated restingCD4⁺ T cells to reflect the diversity of virus that may be encounteredin vivo following reactivation of latent virus. Despite the diversity ofthe AR virus isolates, DART activity mirrored that seen withJR-CSF-infected target cells. Modest activity was observed withAR-infected target cells in the absence of CD8⁺ effectors (thusattributed to CD4⁺ T cells; FIG. 16D), with p24 production reduced by0.32 log with 7B2×CD3 and by 0.20 log with A32×CD3 (p=N.S. due to highervariance in response to 7B2×CD3) and by 0.51 log with a 1:1 cocktail ofboth DARTs (p<0.05), whereas no activity was observed with the controlDARTs (FIG. 16D). The addition of HIV×CD3 DARTs to a mixture of ARvirus-infected CD4+ target cells and autologous CD8+ effector cells ledto significantly enhanced reductions in p24 production. At an E:T ratioof 1:10, p24 production was reduced by 0.51 log with 7B2×CD3 (p<0.05),by 0.37 log with A32×CD3 and by 0.79 log with a 1:1 cocktail of the two(p<0.05), compared to a reduction of only 0.02 log with CD8⁺ cells alone(FIG. 16E). A trend towards decreased p24 production in the presence ofHIV×CD3 DARTs was also seen at the higher E:T ratio of 1:1, although themagnitude of the effect was reduced by the variable baseline CD8⁺activity seen in the absence of DARTs (FIG. 16F). Notably, ex vivo DARTactivity was observed with lymphocytes from all 5 patients evaluatedwith at least one of the two HIV×CD3 DARTs, and in all cases with the1:1 DART cocktail.

HIV×CD3 DARTs Redirect T Cells from HIV-Infected Individuals onSuppressive ART to Clear Virus from Resting CD4′ T Cells FollowingInduction of Latent Virus Expression.

Ultimately, a reagent used in the “shock and kill” HIV eradicationstrategy must recognize and clear rare infected cells that are likely toexpress low levels of antigen as they emerge from latency. A latencyclearance assay as previously described (49) was employed. This assayseeks to measure the ability of DARTs to redirect autologous CD8⁺ Tcells to reduce viral recovery following induction of resting CD4⁺ Tcells of HIV-infected individuals on suppressive ART. Addition of fullyactive DARTs or a 1:1 cocktail of A32×CD3 and 7B2×CD3 to a co-culture ofCD8⁺ T cells with PHA-stimulated resting CD4⁺ T cells at an E:T ratio of1:10 reduced viral recovery in all 6 out of 6 patients, although themagnitude of reduction varied amongst patients. (FIGS. 17A, 22).

Reversal of HIV latency using maximal mitogen stimulation in vivo is notclinically practical (50). However, the presentation of viral antigenfollowing the reversal of latency with agents that do not result inglobal T cell activation, such as vorinostat (VOR), may be less robustthan that following maximal mitogen stimulation. To evaluate the HIV×CD3DARTs in a clinically relevant context, a physiologically relevantexposure to VOR that models that obtained following a single 400 mg invivo dose (18) to induce latent viral envelope expression was used. Inthis setting, addition of CD8⁺ cells at an E:T ratio of 1:10 plus fullyactive DARTs led to a reduction in viral recovery following a 24 hourco-culture period when compared to CD8⁺ cells without or with controlDARTs in 4 of 5 patients tested. In the single patient who did notrespond to DARTs after a 24 hour co-culture period (patient 795),extending the co-culture period from 24 hours to 96 hours led tocomplete ablation of viral recovery (FIGS. 17B, 22).

Discussion

Significant hurdles in the elimination of the latent HIV-1 reservoirinclude: 1) the limited ability of the immune system to recognize rareHIV-1 infected cells presenting modest levels of HIV antigen prior to orfollowing induction with latency reversing agents (LRA) (38, 51); 2) thepresence of CD8⁺ cytotoxic T lymphocyte escape mutants in the HIV-1latent reservoir (52); and 3) the low frequency of circulatingHIV-specific CD8⁺ T cells in patients on ART and the necessity toactivate them due to inadequate stimuli provided by infected cells (38).Described herein are data that HIV×CD3 DARTs could overcome each ofthese major obstacles.

HIV×CD3 DARTs with HIV arms derived from the non-neutralizing mAbs A32and 7B2 were able to recognize HIV-1 Env-expressing cell lines and toelicit redirected T-cell killing activity, even when cell surface Envexpression appeared low. In addition, HIV×CD3 DARTs were effective exvivo in redirecting CD8⁺ T cells to clear resting CD4⁺ T cells obtainedfrom aviremic, ART-treated patients following exposure to VOR.

HIV-1 isolates represented in the latent reservoir are reported toinclude escape mutants generated by the CD8⁺ T cell responses (52),which may limit the ability of the MHC class I-restricted CD8⁺ CTLresponses induced by natural infection to clear HIV-1 infected cells.The A32 and 7B2 arms of the HIV×CD3 DARTs are based on broadly reactivenon-neutralizing anti-HIV mAbs that interact with highly conservedresidues in gp120 and gp41, respectively, and efficiently mediate ADCCactivity against cells infected with HIV-1 isolates of various subtypes.Of note, the A32 mAb epitope is the earliest one known to be expressedon the surface of infected cells during the syncytia-formation process(53) or following tier 2 virus infection (54) and the 7B2 mAb epitope isaccessible on gp41 stumps, which are expressed on the surface ofinfected cells during budding and retained at the membrane surface whengp120 subunits dissociate (29, 55). These properties are indicative ofthe accessibility of the A32 and 7B2 epitopes on the surface of infectedcells. Importantly, the existence of CTL escape mutants is not alimitation, because CTL epitopes are irrelevant to DART-mediatedredirected killing activity. Further, effector T-cells recruited bybispecific molecules like DARTs are polyclonal and not MHC-restricted(33). Consistent with these assertions, A32×CD3 and 7B2×CD3 wereeffective at redirecting CD8⁺ T cells from patients to clear CD4⁺ cellsinfected by their own autologous reservoir (AR) virus, regardless of thepresence of any escape mutations that may have accumulated beforeinitiation of therapy (52). Interestingly, upon in vitro activation ofthe CD4⁺ T cells used as target cells, a specific reduction in virusrecovery in absence of CD8⁺ T cells was observed, suggesting that DARTscould also recruit cytotoxic CD4⁺ T cells under these particularexperimental conditions. In line with these, it was found that DARTsinduced activation of CD4⁺ T cells in the presence of Env expressingJurkat-522 F/Y cells, and were capable of increasing degranulation ofCD4⁺ T cells when co-cultured with infected autologous target cells fromHIV positive individuals. Cytotoxic CD4⁺ T cells have been previouslyreported in the context of responses to HIV-1 (56) and Cytomegalovirus(57). Further studies will be necessary to determine whether effectiveDART recruitment and redirection of cytotoxic CD4⁺ T cells occurs underin vivo settings.

The relative potencies of the A32×CD3 and 7B2×CD3 DARTs varied among thedifferent test systems employed in our studies, most likely due tovariations in the characteristics of the Env-expressing target cellsand/or effector T-cells. However, whenever one of the DARTs exhibitedgreater activity than the other, activity similar to that of the morepotent DART when combinations of the two DARTs were utilized in thestudies with infected patients' cells (FIGS. 16A-16H and 17A-17B) wasconsistently observed. Thus, combinations of DARTs targeting differentHIV epitopes may be an advantageous strategy to maximize both level andbreadth of activity, similar to what has been described for combinationsof ADCC-mediating (58) or broadly neutralizing anti-HIV-1 mAbs (59, 60).

Eliminating the pool of latently infected cells by HIV-1-specific CD8⁺ Tcell responses is limited by the low frequency of these cells ininfected individuals and the need to activate them from the restingstate (38). With resting CD8⁺ T cells from HIV-1 seronegativeindividuals lacking any previous exposure to HIV-1 antigens, HIV×CD3DARTs induced degranulation of up to 23% of these resting CD8⁺ T cellswhen incubated with the autologous HIV-1 infected target cells destinedto be killed. DARTs were also capable of redirecting CD8⁺ T cells fromHIV-1 seropositive individuals who received antiretroviral therapy inviral clearance assays. Therefore, HIV×CD3 DART proteins can effectivelyrecruit and redirect CD8⁺ T cytotoxic cells independent of previousexposure to HIV antigens, and regardless of any functional impairmentthat may remain in chronic HIV-1 infection (46, 47, 61).

DART redirected T cell activity against HIV-1 Env-expressing targets wasdependent on HIV×CD3 DART concentration, effector:target (E:T) cellratio and incubation time. The monovalent nature of each of the bindingarms of the HIV×CD3 DART molecule ensures that target cell killingdepends exclusively upon effector/target cell co-engagement, as has beenobserved with CD19×CD3 and other DARTs (31, 32, 34). No HIV×CD3DART-mediated T-cell activation or redirected killing activity wasobserved in the absence of Env expression on target cells. Similarly,with T-cells from HIV-infected patients on suppressive ART, no T-cellactivation was observed in the absence of virus-infected target cells.Because they should elicit cytotoxic activity from circulating T cellsonly in the proximity of HIV-1 infected Env-expressing target cells,HIV×CD3 DARTs are not expected to elicit widespread systemic effects,such as inflammatory cytokine release, in HIV-infected patients on ARTdue to the scarcity of the Env-expressing target cells. The specificityof T-cell redirected responses elicited by HIV×CD3 DARTs will be ofcritical importance clinically, considering that HIV infection inducesnonspecific activation of the immune system in both the acute andchronic phases of the disease, in HIV-1 specific T-cell subsets as wellas in general CD8+ T cell populations (62-64).

HIV-infected CD4⁺ T cells expressing cell surface Env are the primary invivo targets for HIV×CD3 DART-redirected T cell killing activity.Because these target cells also express CD3, the DART molecules couldmediate synapses between infected and uninfected CD4⁺ T cells that,rather than or in addition to redirecting the killing of infected cells,conceivably could facilitate the spread of virus to uninfected cells.However, no evidence to suggest that DARTs enhanced the spread of viruswas observed, as DARTs reduced p24 production even in the absence ofCD8+ T cells (FIGS. 16A and 16D).

In summary, the experiments described herein demonstrate that HIV×CD3DARTs, with HIV arms derived from the non-neutralizing A32 and 7B2 mAbs,are specific and potent agents to redirect cytolytic T-cells againsttarget cells consisting of 1) HIV-1 Env-expressing CD4⁺ cell lines, 2)activated CD4⁺ cells from seronegative individuals infected with HIV-1IMCs of different subtypes, 3) activated CD4⁺ cells from seropositivepatients on suppressive ART infected with JR-CSF or autologous reservoirvirus, or 4) resting CD4⁺ cells from HIV-infected patients exposed exvivo to a T-cell mitogen (phytohemagglutinin, PHA) or latency reversingagent (vorinostat, VOR). Importantly, the studies demonstrated thatautologous CD8⁺ T cells from HIV-infected patients on suppressive ARTwere efficacious as effector cells in the presence of DARTs. Thedemonstration of HIV×CD3 DART-mediated T cell killing activity in thepresence of vorinostat is particularly notable because it providesevidence of activity against authentic latent virus isolates expressedfrom HIV-infected patients' cells in a model system designed to mimicpotential clinical HIV eradication strategies, similar to earlierfindings using ex-vivo expanded CTLs (49). Thus, the disclosed dataindicate that HIV×CD3 DARTs are suitable agents for testing in vivo incombination with LRAs in “shock and kill” HIV eradication strategies.

Methods

We have reanalyzed the data using the Dunnett's test for multiplecomparisons deemed appropriate due of the relative limited number ofsamples in our studies. The calculated p values are now indicated in themain text (page 14) and in the legends for FIGS. 5-7. The Methodssection for the statistical analyses has also been revised.

Patient Population.

Leukapheresis samples were obtained from HIV seronegative donors orHIV-infected donors with undetectable plasma viremia (<50 copies/mL) onstable ART for at least 6 months, as indicated. Written informed consentwas obtained from each patient and the study was approved by the Dukeand UNC Biomedical Institutional Review Boards.

Infectious Molecular Clones (IMCs).

HIV-1 IMCs for subtype B BaL, subtype AE CM235 and subtype C 1086.0 weregenerated with the backbone derived from NHL4-3 isolate as previouslydescribed (65, 66). All IMCs expressed the Renilla luciferase reportergene and preserved all nine viral open reading frames. The Renillaluciferase reporter gene was expressed under the control of the HIV-1Tat gene. Upon HIV-1 infection of CD4+ T cells, expression of Tat duringHIV-1 replication will induce luciferase expression, which allowsquantitation of infected cells by measuring relative luminescence units(RLU).

Construction, Expression, and Purification of HIV×CD3 DARTs.

The DARTs were produced from plasmids that coexpressed two polypeptidechains: one with VL of anti-CD3 linked to VH of anti-HIV; the secondwith VL of anti-HIV linked to VH of anti-CD3. The carboxy termini of thetwo polypeptide chains consist of paired oppositely chargedE-coil/K-coil dimerization domains, which include an interchaindisulfide bond (FIGS. 10A-10C). The HIV arm sequences were derived fromthe non-neutralizing mAbs, A32 [Genbank accession numbers 3TNM_H and3TNM_L] and 7B2 [Genbank accession numbers AFQ31502 and AFQ31503], andthe CD3 arm sequence was derived from hXR32, a humanized mouseanti-human CD36 mAb (L. Huang, L. S. Johnson, CD3-binding moleculescapable of binding to human and nonhuman CD3, U.S. Patent. 20140099318(2014)). Control DARTs were similarly constructed by replacing eitherthe HIV or CD3 specificity with an irrelevant specificity from ananti-fluorescein mAb (4420) (67) or anti-RSV mAb (palivizumab) (68).DART-encoding sequences were cloned into CET1019AD UCOE vectors (EMDMillipore), transfected into CHO cells and proteins purified asdescribed previously (31). Purified proteins were analyzed by SDS-PAGE(NuPAGE Bis-Tris gel system, Invitrogen) and analytical SEC (TSKGS3000SW×L SE-HPLC, Tosoh Bioscience).

ELISA.

For monospecific binding assays, a MaxiSorp microtiter plate (Nunc)coated with recombinant proteins (human CD36/8 heterodimer, JR-FLgp140ΔCF; (69)) in bicarbonate buffer was blocked with 3% BSA and 0.1%Tween-20. DART proteins were applied, followed by sequential addition ofbiotinylated anti-EK coil antibody and streptavidin-HRP (BDBiosciences). For bispecific binding assays, the plate was coated withJRFL gp140ΔCF and DART application was followed by sequential additionof biotinylated CD3ε/δ and streptavidin-HRP. HRP activity was detectedwith SuperSignal ELISA Pico chemiluminescent substrate (ThermoScientific).

SPR Analysis.

HIV×CD3 DART binding to antigens was analyzed by BIAcore 3000 biosensor(GE, Healthcare) as previously described (31, 32). Human CD3ε/δ wasimmobilized on the CMS sensor chip according to the manufacturer'sprocedure. DART binding to immobilized CD3 was analyzed to assess theproperties of the CD3 arm and HIV-1 Env protein binding to HIV DARTcaptured on immobilized CD3 was analyzed to assess the properties of theHIV arm. JRFL gp140ΔCF was used to assess 7B2×CD3 binding and M.ConSgp140ΔCFI (69) was used to assess A32×CD3 binding. The different Envproteins were utilized because A32×CD3 did not bind efficiently to JR-FLgp140ΔCF and M.ConS gp140ΔCFI lacks the gp41 binding site for 7B2×CD3.Binding experiments were performed in 10 mM HEPES, pH 7.4, 150 mM NaCl,3 mM EDTA and 0.005% P20 surfactant. Regeneration of immobilizedreceptor surfaces was performed by pulse injection of 10 mM glycine, pH1.5. K_(D) values were determined by a global fit of binding curves tothe Langmuir 1:1 binding model (BIA evaluation software v4.1).

Cell Lines.

Jurkat-522 F/Y GF cells, which constitutively express a fusion proteinof Copepod Green Fluorescent Protein (copGFP) and Firefly Luciferase(System Biosciences), were generated at Macrogenics from Jurkat-522 F/Ycells by transduction and clone selection. HEK293-D371 cells, which havedoxycycline-inducible expression of HIV-1 CM244 (subtype AE) gp140, wereobtained from Dr. John Kappes (University of Alabama at Birmingham).

Flow Cytometric Analysis of DART or mAb Binding to Cells.

DARTs at 4 μg/mL were incubated with 10⁵ cells in 200 μL FACS buffercontaining 10% human AB serum for 30 minutes at room temperature. Afterwashing, cells were resuspended in 100 μL of 1 μg/mL biotin-conjugatedmouse anti-EK antibody (recognizes the E/K heterodimerization region ofDART proteins), mixed with 1:500 diluted streptavidin-PE and incubatedin the dark for 45 minutes at 2-8° C. Cells were washed, resuspendedwith FACS buffer, and analyzed with a BD Calibur flow cytometer andFlowJo software (TreeStar, Ashland Oreg.). Binding to IMC-infected CD4⁺T cells from normal human donors was conducted as previously described(54) for the A32 and 7B2 mAbs, and with biotin-conjugated mouse anti-EKantibody and 1:500 diluted streptavidin-PE for the HIV×4420 DARTs.

Redirected T-Cell Cytotoxicity Assay Against HIV-1 Env-Expressing CellLines and Assessment of T-Cell Activation.

Pan T cells were isolated from healthy human PBMCs with the Dynabeads®Untouched™ Human T Cells Kit (Invitrogen). HIV-1 Env expressing celllines (1-4×10⁵ cells/mL) were treated with serial dilutions of DARTs,together with human T cells at an effector:target (E:T) ratio=10:1, orotherwise at varying E:T ratios as indicated, and incubated at 37° C.,5% CO₂ overnight. Cytotoxicity was measured by lactate dehydrogenase(LDH) release (CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega)as described previously (32). With the Jurkat-522 F/Y GF cell line,cytotoxicity was also measured by luminescence using Luciferase-Glosubstrate (Promega). Specific lysis was calculated from luminiscencecounts (RLU): cytotoxicity (%)=100×(1−(RLU of Sample÷RLU of Control)),where Control=average RLU of target cells incubated with effector cellsin the absence of DART. Data were fit to a sigmoidal dose-responsefunction to obtain 50% effective concentration (EC₅₀) and percentmaximum specific lysis values. T-cell activation was measured by FACSanalysis after cells in the assay plate were labeled with CD8-FITC,CD4-APC, and CD25-PE antibodies (BD Biosciences), followed by cellcollection by FACS Calibur flow cytometer equipped with acquisitionsoftware CellQuest Pro Version 5.2.1 (BD Biosciences). Data analysis wasperformed using FlowJo software (Treestar, Inc).

Redirected T-Cell Cytotoxicity Assay Against HIV-1 IMC-Infected CD4⁺Cells.

Cryopreserved resting PBMC from normal healthy HIV-1 seronegative donorswere activated for 72 hours with anti-human CD3 (clone OKT3;eBioscience) and anti-human CD28 (clone CD28.2; BD Pharmingen).Subsequently, a CD4⁺ enriched cell population (purity>92.3%;average±standard deviation 95.73±2.6%) was obtained by depletion of CD8⁺T cells using magnetic beads (Miltenyi Biosciences), spinoculated inpresence of the luciferase-expressing IMC representing HIV-1 subtype AE(CM235), B (BaL) or C (1086.C) and cultured for 72 hours. CD4⁺ infectedtarget cells were incubated with resting CD8⁺ effector cells (isolatedby negative selection from autologous PBMC, CD8⁺ T cell Isolation Kit,Miltenyi Biosciences) at 33:1, 11:1, 3:1, and 0:1 E:T ratios in theabsence or presence of DARTs for 6-48 hours at concentration rangingfrom 1,000 to 0.0001 ng/mL. Uninfected and infected target cells alonewere included as additional controls. Each condition was tested induplicate. After incubation, ViviRen™ Live Cell Substrate (Promega) wasadded and RLU measured on a luminometer; percentage specific lysis (%SL) of target cells was determined as described previously (58).

T-Cell Degranulation (CD107) Assay.

As described for the cytotoxicity assay with HIV-1 IMC-infected cells astargets, activated CD4⁺ cells infected with HIV-1 BaL IMC were platedwith resting CD8⁺ effector cells at a 33:1 E:T ratio in the absence orpresence of 1 ng/mL DARTs and incubated for 6 hour. For the CD4 T celldegranulation, activated CD4⁺ T cells were either infected with JR-CSFand labeled with the viability (NFL1) and target specific (TFL4) markersroutinely utilized in our ADCC assay (70) or added to targets aseffectors at a 10:1 ratio prior to addition of DARTs. Each condition wastested in duplicate. CD107 PE-Cy5 (clone H4A3; eBioscience) was titeredand added during the last six hours of the incubation along withMonensin solution (BD GolgiStop) (71). A panel of antibodies consistingof LIVE/DEAD Aqua stain, anti-CD3 APC-H7 (clone SK7; BD Pharmingen),anti-CD4 BV605 (clone OKT4; Biolegend), anti-CD8 BV650 (clone RPA-T8;Biolegend) was used to detect CD107⁺ CD8⁺ T cells. After washing andfixation, samples were acquired on a custom made LSRII (BD Bioscience,San Jose, Calif.) within the next 24 hours. A minimum of 300,000 totalviable events was acquired for each test. The analysis of the data wasperformed using the Flow-Jo software (Treestar, Ashland, Oreg.).

T-Cell Viability and Activation Assays.

CD8⁺ T cells and CD8 depleted PBMCs obtained from HIV infected ARTsuppressed patients were plated at 5×10⁴ cells per well in 96 wellplates with 100 ng/mL of the indicated DART. Cells were cultured in 0.2mL of cIMDM media supplemented with 10% FBS, 1% Penicillin/Streptomycinand 5U/mL IL-2 for 7 days, and then stained with the followingantibodies: HLA-DR-PerCP (clone L243), CD25-PE (clone M-A251), CD8-FITC(clone HIT8a), CD8-PE (clone HIT8a), CD4-FITC (clone RPA-T4), andAnnexin V-PE and 7-AAD (all BD biosciences, San Jose, Calif.).

Redirected T-Cell Viral Clearance Assay.

CD8⁺ T-cells were isolated from PBMCs by positive selection (EasySephuman CD8⁺ Selection Kit, Stem Cell). CD8-depleted PBMCs were firstactivated with 2 μg/mL of PHA (Remel, Lenexa, Kans.) and 60U/mL of IL-2,and then infected by spinoculation at 1200×g for 90 minutes with eitherJR-CSF or autologous reservoir virus (AR) at an MOI of 0.01 aspreviously described (47). AR virus was obtained from pooledsupernatants of replicate wells from outgrowth assays of resting CD4+T-cells for each patient performed as previously described (72).Fifty-thousand (5×10⁴) targets/well were co-cultured with CD8⁺ T cellsin triplicate at the indicated E:T ratio in the absence or presence of100 ng/mL of DART in 0.2m of cIMDM media supplemented with 10% FBS, 1%Penicillin/Streptomycin and 5 U/mL IL-2. For experiments performed inthe presence of antiretrovirals (ARVs), 24 hours after spinoculationcells were washed and 104 of raltegravir and 404 of abacavir were added,and then DARTs and CD8⁺ T-cells were added to cultures. Supernatant wasassayed on day 7 by p24 ELISA (ABL, Rockville, Md.). Results arecalculated as the log (p24 of infected target cells only control dividedby p24 of the test condition).

Latency Clearance Assay (LCA).

The reduction of virus recovery from CD4⁺ infected cells was assessed bya standard quantitative viral outgrowth assay using the resting CD4⁺ Tcells of aviremic, ART-treated patients, following the addition ofantiviral effector cells and/or molecules, as previously described (49).In this case the LCA was used to model the ability of DARTs to clearvirus emerging from the latent reservoir under clinically andpharmacologically relevant conditions. Resting CD4⁺ T-cells wereisolated from a leukapheresis product as previously described (72) andexposed to PHA (4 μg/mL) and IL-2 (60U/mL) for 24 hours or vorinostat(VOR) (335 nM, 6 hours) (Merck Research Laboratories), and plated at 0.5to 1×10⁶ cells/well in 12 to 36 replicate wells depending on the size ofthe reservoir. The VOR was then washed off and CD8s added at an E:T of1:10 as well as 100 ng/mL of the indicated DART. Cells were co-culturedfor 24 hours (unless specified otherwise) following which the DARTproteins were washed off and allogeneic CD8-depleted PBMCs from an HIVnegative donor were added to amplify residual virus. Supernatant wasassayed for the presence of p24 antigen on day 15 for each well. Resultsare calculated as % viral recovery [(# of positive wells/total numberplated)×100], normalized to a control in which no CD8⁺ T cells areadded.

Statistical Analysis.

Statistical comparisons between groups were analyzed using the Dunnett'stest for multiple comparisons using GraphPad Prism Softward (La Jolla,Calif.); p values<0.05, calculated with Dunnett correction for multiplecomparisons, were considered significant. Dunnett's test for multiplecomparisons was deemed appropriate due to the relative limited number ofsamples in the studies.

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1.-32. (canceled)
 33. A bispecific molecule comprising a firstpolypeptide chain and a second polypeptide chain covalently bonded toone another, wherein: (I) the first polypeptide chain comprises in theN- to C-terminal direction: (i) a domain (A) comprising a binding regionof the light chain variable domain of a first immunoglobulin (VL1)comprising the VL CDR3, CDR2 and CDR1 of HIV-1 antibody A32 (SEQ IDNO:78) or 7B2 (SEQ ID NO:55); (ii) a domain (B) comprising a bindingregion of a heavy chain variable domain of a second immunoglobulin (VH2)comprising the VH CDR3, CDR2 and CDR1 of an antibody specific for anepitope of CD3 or CD16, wherein domains (A) and (B) are separated fromone another by a peptide linker 1; and (iii) a domain (C) comprising aheterodimer promoting domain including a K coil or E coil; wherein theheterodimer promoting domain (C) and domain (B) are separated by apeptide linker 2; (II) the second polypeptide chain comprises in the N-to C-terminal direction: (i) a domain (D) comprising a binding region ofa light chain variable domain of the second immunoglobulin (VL2)comprising the VL CDR3, CDR2 and CDR1 of an antibody specific for theepitope of CD3 or CD16; (ii) a domain (E) comprising a binding region ofa heavy chain variable domain of the first immunoglobulin (VH1)comprising the VH CDR3, CDR2, and CDR1 of HIV-1 antibody A32 (SEQ IDNO:77), or 7B2 (SEQ ID NO:56), wherein domains (D) and (E) are separatedfrom one another by a peptide linker 1; and (iii) a domain (F)comprising a heterodimer promoting domain including a K coil or E coil;wherein the heterodimer promoting domain (F) and domain (E) areseparated by a peptide linker 2, and wherein: the domains (A) and (B) donot associate with one another to form an epitope binding site; thedomains (D) and (E) do not associate with one another to form an epitopebinding site; the domains (A) and (E) associate to form a binding sitethat binds the HIV-1 envelope like the A32 or 7B2 antibody (1); thedomains (B) and (D) associate to form a binding site that binds theepitope of CD3 or CD16; the K coil comprises residues 240-267 of SEQ IDNO: 19, and the E coil comprises residues 249-276 of SEQ ID NO: 17; andpeptide linker 2 comprises residues 244-248 of SEQ ID NO:
 17. 34. Thebispecific molecule of claim 33, wherein (i) the VL1 comprises the VLCDR3, CDR2, and CDR1 of HIV-1 antibody A32 (SEQ ID NO: 78); (ii) the VH1comprises the VH CDR3, CDR2, and CDR1 of HIV-1 antibody A32 (SEQ ID NO:77); and (iii) the domains (B) and (D) associate to form a binding sitethat binds the epitope of CD3.
 35. The bispecific molecule of claim 33,wherein (i) the VL1 comprises the VL CDR3, CDR2, and CDR1 of HIV-1antibody A32 (SEQ ID NO: 78); (ii) the VH1 comprises the VH CDR3, CDR2,and CDR1 of HIV-1 antibody A32 (SEQ ID NO: 77); and (iii) the domains(B) and (D) associate to form a binding site that binds the epitope ofCD16.
 36. The bispecific molecule of claim 33, wherein (i) the VL1comprises the VL CDR3, CDR2, and CDR1 of HIV-1 antibody 7B2 (SEQ ID NO:55); (ii) the VH1 comprises the VH CDR3, CDR2, and CDR1 of HIV-1antibody 7B2 (SEQ ID NO: 56); and (iii) the domains (B) and (D)associate to form a binding site that binds the epitope of CD3.
 37. Thebispecific molecule of claim 33, wherein (i) the VL1 comprises the VLCDR3, CDR2, and CDR1 of HIV-1 antibody 7B2 (SEQ ID NO: 55); (ii) the VH1comprises the VH CDR3, CDR2, and CDR1 of HIV-1 antibody 7B2 (SEQ ID NO:56); and (iii) the domains (B) and (D) associate to form a binding sitethat binds the epitope of CD16.
 38. The bispecific molecule of claim 33,wherein (i) the VL1 comprises the VL CDR3, CDR2, and CDR1 of HIV-1antibody A32 (SEQ ID NO: 78); (ii) the VH1 comprises the VH CDR3, CDR2,and CDR1 of HIV-1 antibody A32 (SEQ ID NO: 77); (iii) the VL2 comprisesthe VL CDR3, CDR2, and CDR1 of anti-CD3 antibody (SEQ ID NO: 52); and(iv) the VH2 comprises the VH CDR3, CDR2, and CDR1 of anti-CD3 antibody(SEQ ID NO: 51).
 39. The bispecific molecule of claim 33, wherein (i)the VL1 comprises the VL CDR3, CDR2, and CDR1 of HIV-1 antibody A32 (SEQID NO: 78); (ii) the VH1 comprises the VH CDR3, CDR2, and CDR1 of HIV-1antibody A32 (SEQ ID NO: 77); (iii) the VL2 comprises the VL CDR3, CDR2,and CDR1 of anti-CD16 antibody (SEQ ID NO: 54); and (iv) the VH2comprises the VH CDR3, CDR2, and CDR1 of anti-CD16 antibody (SEQ ID NO:53).
 40. The bispecific molecule of claim 33, wherein (i) the VL1comprises the VL CDR3, CDR2, and CDR1 of HIV-1 antibody 7B2 (SEQ ID NO:55); (ii) the VH1 comprises the VH CDR3, CDR2, and CDR1 of HIV-1antibody 7B2 (SEQ ID NO: 56); (iii) the VL2 comprises the VL CDR3, CDR2,and CDR1 of anti-CD3 antibody (SEQ ID NO: 52); and (iv) the VH2comprises the VH CDR3, CDR2, and CDR1 of anti-CD3 antibody (SEQ ID NO:51).
 41. The bispecific molecule of claim 33, wherein (i) the VL1comprises the VL CDR3, CDR2, and CDR1 of HIV-1 antibody 7B2 (SEQ ID NO:55); (ii) the VH1 comprises the VH CDR3, CDR2, and CDR1 of HIV-1antibody 7B2 (SEQ ID NO: 56); (iii) the VL2 comprises the VL CDR3, CDR2,and CDR1 of anti-CD16 antibody (SEQ ID NO: 54); and (iv) the VH2comprises the VH CDR3, CDR2, and CDR1 of anti-CD16 antibody (SEQ ID NO:53).
 42. The bispecific molecule of claim 33, wherein the domain (A)comprises VL of HIV-1 antibody A32 (SEQ ID NO: 78); the domain (E)comprises VH of HIV-1 antibody A32 (SEQ ID NO: 77); and the domains (B)and (D) associate to form a binding site that binds the epitope of CD3.43. The bispecific molecule of claim 33, wherein the domain (A)comprises VL of HIV-1 antibody A32 (SEQ ID NO: 78); the domain (E)comprises VH of HIV-1 antibody A32 (SEQ ID NO: 77); the domain (B)comprises VH of anti-CD3 antibody (SEQ ID NO: 51); and the domain (D)comprises VL of anti-CD3 antibody (SEQ ID NO: 52).
 44. The bispecificmolecule of claim 33, wherein the domain (A) comprises VL of HIV-1antibody A32 (SEQ ID NO: 78); the domain (E) comprises VH of HIV-1antibody A32 (residues 119-241 of SEQ ID NO: 11); and the domains (B)and (D) associate to form a binding site that binds the epitope of CD3.45. The bispecific molecule of claim 33, wherein the domain (A)comprises VL of HIV-1 antibody A32 (SEQ ID NO: 78); the domain (E)comprises VH of HIV-1 antibody A32 (residues 119-241 of SEQ ID NO: 11);the domain (B) comprises VH of anti-CD3 antibody (SEQ ID NO: 51); andthe domain (D) comprises VL of anti-CD3 antibody (SEQ ID NO: 52). 46.The bispecific molecule of claim 33, wherein the domain (A) comprises VLof HIV-1 antibody 7B2 (SEQ ID NO: 55); the domain (E) comprises VH ofHIV-1 antibody 7B2 (SEQ ID NO: 56), and the domains (B) and (D)associate to form a binding site that binds the epitope of CD3.
 47. Thebispecific molecule of claim 33, wherein the domain (A) comprises VL ofHIV-1 antibody 7B2 (SEQ ID NO: 55); the domain (E) comprises VH of HIV-1antibody 7B2 (SEQ ID NO: 56), and the domain (B) comprises VH ofanti-CD3 antibody (SEQ ID NO: 51); and the domain (D) comprises VL ofanti-CD3 antibody (SEQ ID NO: 52).
 48. The bispecific molecule of claim33, wherein the domain (A) comprises VL of HIV-1 antibody A32 (SEQ IDNO: 78); the domain (E) comprises VH of HIV-1 antibody A32 (SEQ ID NO:77), and the domains (B) and (D) associate to form a binding site thatbinds the epitope of CD16.
 49. The bispecific molecule of claim 33,wherein the domain (A) comprises VL of HIV-1 antibody A32 (SEQ ID NO:78); the domain (E) comprises VH of HIV-1 antibody A32 (SEQ ID NO: 77);the domain (B) comprises VH of anti-CD16 antibody (SEQ ID NO: 53); andthe domain (D) comprises VL of anti-CD16 antibody (SEQ ID NO: 54). 50.The bispecific molecule of claim 33, wherein the domain (A) comprises VLof HIV-1 antibody A32 (SEQ ID NO: 78); the domain (E) comprises VH ofHIV-1 antibody A32 (residues 119-241 of SEQ ID NO: 11), and the domains(B) and (D) associate to form a binding site that binds the epitope ofCD16.
 51. The bispecific molecule of claim 33, wherein the domain (A)comprises VL of HIV-1 antibody A32 (SEQ ID NO: 78); the domain (E)comprises VH of HIV-1 antibody A32 (residues 119-241 of SEQ ID NO: 11);the domain (B) comprises VH of anti-CD16 antibody (SEQ ID NO: 53); andthe domain (D) comprises VL of anti-CD16 antibody (SEQ ID NO: 54). 52.The bispecific molecule of claim 33, wherein the domain (A) comprises VLof HIV-1 antibody 7B2 (SEQ ID NO: 55); the domain (E) comprises VH ofHIV-1 antibody 7B2 SEQ (ID NO: 56), and the domains (B) and (D)associate to form a binding site that binds the epitope of CD16.
 53. Thebispecific molecule of claim 33, wherein the domain (A) comprises VL ofHIV-1 antibody 7B2 (SEQ ID NO: 55); the domain (E) comprises VH of HIV-1antibody 7B2 SEQ (ID NO: 56); the domain (B) comprises VH of anti-CD16antibody (SEQ ID NO: 53); and the domain (D) comprises VL of anti-CD16antibody (SEQ ID NO: 54).
 54. The bispecific molecule of claim 38, 39,40, or 41, wherein the bispecific molecule further comprises anFc-domain.
 55. The bispecific molecule of claim 38 or 40, wherein: thefirst polypeptide chain further comprises a CH2-CH3 domain, wherein theCH2-CH3 domain and domain (C) are separated by a peptide linker 3 or aspacer-linker 3; (ii) the bispecific molecule further comprises a thirdpolypeptide chain comprising in the N- to C-terminal direction a peptidelinker 3 and a CH2-CH3 domain; and (iii) the CH2-CH3 domains of thefirst and third polypeptide form the Fc domain.
 56. The bispecificmolecule of claim 55, wherein: (i) the CH2-CH3 domain of the firstpolypeptide chain comprises SEQ ID NO: 42 and the CH2-CH3 domain of thethird polypeptide chain comprises SEQ ID NO: 43; or (ii) the CH2-CH3domain of the first polypeptide chain comprises SEQ ID NO: 43 and theCH2-CH3 domain of the third polypeptide chain comprises SEQ ID NO: 42.57. A composition comprising the bispecific molecule of claim 38 or 40.58. The composition of claim 57, further comprising a second bispecificmolecule comprising a first arm with the binding specificity of HIV-1antibody A32, HIV-1 antibody 7B2, HIV-1 antibody CH28, or HIV-1 antibodyCH44 and a second arm targeting CD3 or CD16, wherein the first andsecond bispecific molecules are different.
 59. A composition comprisingthe bispecific molecule of claim
 54. 60. The composition of claim 59,further comprising a second bispecific molecule comprising a first armwith the binding specificity of HIV-1 antibody A32, HIV-1 antibody 7B2,HIV-1 antibody CH28, or HIV-1 antibody CH44 and a second arm targetingCD3 or CD16, wherein the first and second bispecific molecules aredifferent.
 61. A composition comprising the bispecific molecule of claim55.
 62. The composition of claim 61, further comprising a secondbispecific molecule comprising a first arm with the binding specificityof HIV-1 antibody A32, HIV-1 antibody 7B2, HIV-1 antibody CH28, or HIV-1antibody CH44 and a second arm targeting CD3 or CD16, wherein the firstand second bispecific molecules are different.
 63. A method to treatHIV-1 infection in a subject in need thereof comprising administering tothe subject a composition comprising the bispecific molecule of claim 38or 40 in a therapeutically effective amount.
 64. The method of claim 63,further comprising administering a latency activating agent.
 65. Themethod of claim 64, wherein the latency activating agent is vorinostat,romidepsin, panobinostat, disulfiram, JQ1, bryostatin, PMA, inonomycin,or any combination thereof.
 66. A method to treat HIV-1 infection in asubject in need thereof comprising administering to the subject acomposition comprising the bispecific molecule of claim 54 in atherapeutically effective amount.
 67. The method of claim 66, furthercomprising administering a latency activating agent.
 68. The method ofclaim 67, wherein the latency activating agent is vorinostat,romidepsin, panobinostat, disulfiram, JQ1, bryostatin, PMA, inonomycin,or any combination thereof.
 69. A vector comprising nucleic acidcomprising nucleotides encoding the bispecific molecule of claim
 33. 70.A composition comprising a vector comprising a nucleic acid encoding thebispecific molecule of claim 33.